Inhibition and enhancement of reprogramming by chromatin modifying enzymes

ABSTRACT

In one aspect, the disclosure provides methods and compositions for the production of stem cells. In one aspect, the disclosure provides methods and uses of stem cells. In some embodiments, the stem cells are induced pluripotent stem cells.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application Ser. No. 61/547,404 filed Oct. 14, 2011, thedisclosure of which is incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. R01DK70055 from the National Institutes of Health. The Government hascertain rights in this invention.

FIELD OF THE INVENTION

The field of invention relates to methods and compositions for theproduction of stem cells.

BACKGROUND OF THE INVENTION

A variety of methods are available for reprogramming adult cells toobtain induced pluripotent stem cells. However, the methods currentlyavailable suffer from low efficiency and incomplete programming. Inaddition, some of the methods currently available result in theupregulation of oncogenes, thereby increasing the risk of tumorformation. New methods for the production of induced pluripotent stemcells are needed therefore.

SUMMARY OF THE INVENTION

Aspects of the disclosure provide compositions and methods forreprogramming differentiated cells to produce pluripotent cells (e.g.,stem cells). In some embodiments, certain enzyme inhibitors enhancecellular reprogramming by accelerating the reprogramming process and/orby simplifying the process (for example, by reducing the number and typeof factors required for cellular reprogramming). For example, in someembodiments, methods and compositions described herein can be used toreprogram differentiated cells without using transcription factors thatcan have unwanted side effects such as oncogenesis. In some embodiments,inhibiting one or more of Dot1L (a histone H3 methyltransferase), YY1 (atranscriptional repressor protein), and/or SUV39H1 (a histone-lysineN-methyltransferase) helps promote the reprogramming of differentiatedcells. One or more of these enzymes can be inhibited using anyappropriate technique, including, for example, by inhibiting expression(e.g., transcription, translation, and/or modification) and/or activityof the enzyme(s).

Pluripotent (e.g., induced pluripotent stems cells—iPSCs) producedmethods and compositions described herein are useful for therapeutic andresearch applications.

Accordingly, in some embodiments the disclosure provides methods andcompositions for the production of stem cells. In certain embodiments,the disclosure provides stem cells (for example induced pluripotent stemcells) and methods and compositions for their use.

In some embodiments, a method of producing induced pluripotent stemcells includes inhibiting of Dot1L, YY1, and/or SUV39H1 in adifferentiated cell (e.g., in a preparation containing one or moredifferentiated cell types). In some embodiments, the method alsoincludes culturing the differentiated cell(s) under one or morereprogramming conditions. In some embodiments, the inhibition of Dot1L,YY1, and/or SUV39H1 occurs under the cell reprogramming conditions.However, in some embodiments, inhibition of Dot1L, YY1, and/or SUV39H1is initiated prior to exposing the cell(s) to reprogramming conditions.

In some embodiments, the act of inhibiting includes inhibiting theactivity of Dot1L, YY1, and/or SUV39H1 (e.g., inhibiting themethyltransferase activity of Dot1L). In some embodiments, the activityis inhibited by contacting a differentiated cell with a compositioncomprising one or more enzyme inhibitors (e.g., one or more enzymeinhibitors specific for Dot1L, YY1, and/or SUV39H1).

In some embodiments, the act of inhibiting includes knocking down theexpression of Dot1L, YY1, and/or SUV39H1 (e.g., by inhibitingtranscription and/or translation of the Dot1L, YY1, and/or SUV39H1gene). In some embodiments, a differentiated cell is contacted with oneor more RNAi (e.g., shRNA) molecules that specifically inhibit Dot1L,YY1, and/or SUV39H1 expression.

In some embodiments, the reprogramming conditions include using acocktail containing one or more reprogramming factors (e.g., one or morereprogramming transcription factors). In some embodiments, thereprogramming cocktail includes Oct4 and/or Sox2. In some embodiments,the reprogramming cocktail consists essentially of Oct4 and Sox2. Insome embodiments, the reprogramming cocktail includes Klf4 and/or c-Myc(e.g., in addition to Oct4 and/or Sox2). However, in some embodiments,the reprogramming cocktail does not include Klf4 or c-Myc. In someembodiments, an inhibitor (e.g., a Dot1L inhibitor) is added to thetranscription factor(s) in the reprogramming cocktail.

In some embodiments, the differentiated cell is a somatic cell (e.g., asomatic cell obtained from a subject) or a cultured cell. In someembodiments, the differentiated cell is a fibroblast (e.g., an adultfibroblast). In some embodiments, the differentiated cell is a humancell (e.g., dH1fs, IMR-90 or MRC-5), or a mouse cell.

In some embodiments, the presence of induced pluripotent stem cells isdetermined (e.g., by detecting one or more markers characteristic of aninduced pluripotent stem cell) after cellular reprogramming. In someembodiments; the presence of induced pluripotent stem cells isdetermined by evaluating the presence of one or more markers selectedfrom the group consisting of SSEA4, SSEA3, Tra-1-81, Oct4, Sox2 andNanog. In some embodiments, induced pluripotent stem cells are isolatedafter reprogramming of one or more differentiated cell types.

In some embodiments, the production of induced pluripotent stem cells isaccelerated by inhibiting Dot1L, YY1, and/or SUV39H1 in a differentiatedcell. In some embodiments, the production of induced pluripotent stemcells is more efficient when Dot1L, YY1, and/or SUV39H1 are inhibited ina differentiated cell. Inhibition can occur (e.g., be initiated) before,during, or after culturing the differentiated cell under reprogrammingconditions. A reduction in time and/or increase in efficiency can beobtained relative to a reprogramming of the differentiated cell in whichDot1L, YY1, and/or SUV39H1 are not inhibited.

In some embodiments, a method of producing induced pluripotent stemcells includes upregulating the expression of Nanog and/or Lin28 in adifferentiated cell. In some embodiments, these-cells are cultured(e.g., before, concurrently, and/or subsequently) under reprogrammingconditions to produce induced pluripotent stem cells. In someembodiments, the expression of Nanog and Lin28 is upregulated byinhibiting Dot1L.

In some embodiments, a preparation of induced pluripotent stem cells isprovided wherein the stem cells were produced as described herein. Insome embodiments, a preparation of induced pluripotent stem cellsincludes one or more inhibitors of Dot1L, YY1, and/or SUV39H1 (e.g., oneor more expression and/or activity inhibitors). In some embodiments, theinhibitors are present in trace amounts. In some embodiments, theinduced pluripotent stem cells are human cells.

In some embodiments, the induced pluripotent stems cell candifferentiate (e.g., be differentiated using appropriate factors and/orconditions) into ectoderm, mesoderm and/or endoderm cells.

In some embodiments, a preparation of induced pluripotent stem cells isadministered to a subject. In some embodiments, a preparation ofdifferentiated stem cells (e.g., induced pluripotent stem cells thatwere differentiated ex vivo) is administered to a subject. In someembodiments, the subject is a patient (e.g., a human or animal patient)in need of treatment with pluripotent stem cells and/or differentiatedcells. In some embodiments, a subject in need of treatment is patienthaving brain damage (e.g., associated with a neurodegenerative disordersuch as Parkinson's disease), cancer, spinal cord injury, heart damage,baldness, deafness, diabetes, neuronal defects, blindness, amyotrophiclateral sclerosis, a genetic disorder, infertility, and/or unhealedwounds. It should be appreciated that the pluripotent and differentiatedcell populations described herein may be used in a variety of in vivomethods including but not limited to therapeutic or cosmeticapplications.

In some embodiments, one or more inhibitors of Dot1L, YY1, and/orSUV39H1 is administered to a subject to promote stem cell growth ordevelopment (e.g., locally at or near the site of local administration,or systemically). In some embodiments, one or more inhibitors may beadministered in combination with one or more other factors, includingbut not limited to, one or more transcription factors (e.g., 2, 3, 4 ormore transcription factors such as those described herein), for examplein the form of a transcription factor expressing vector (e.g., forinducing local stem cell populations).

In some aspects, the disclosure provides kits, compositions, and methodsfor identifying enhancers and/or inhibitors of cell reprogramming. Insome aspects, the disclosure provides kits, compositions, and methodsfor producing or isolating induced pluripotent stem cells and/ordifferentiated cells obtained from the stem cells.

These and other aspects and embodiments of the invention are describedin greater detail below.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including”, “comprising”, or “having”,“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures are illustrative only and are not required for enablement ofthe invention disclosed herein.

FIG. 1 illustrates a non-limiting example of a screen for inhibitors andenhancers of cellular reprogramming;

FIG. 2 shows a non-limiting example of cellular reprogramming enhancedby Dot1L inhibition;

FIG. 3 shows a non-limiting example of Dot1L inhibition using a smallmolecule;

FIG. 4 shows a non-limiting example of Nanog and Lin28 associated withreprogramming by Dot1L inhibition;

FIG. 5 shows a non-limiting example of H3K79me2 marks duringreprogramming;

FIG. 6 shows a non-limiting example of knockdown efficiency by shRNA indH1f cells;

FIG. 7 shows a non-limiting example of cell proliferation upon PRC1/2knockdown;

FIG. 8 shows a non-limiting example of Dot1L mRNA and total H3K79me2levels under knock-down conditions;

FIG. 9 shows a non-limiting example of same-well reprogramming ofadmixed control and Dot1L inhibited cell populations;

FIG. 10 shows a non-limiting characterization of shDot1L iPS cells;

FIG. 11 shows a non-limiting example of Dot1L knockdown dynamics duringreprogramming;

FIG. 12 shows a non-limiting example of the Growth dynamics of shDot1Lcells pre- and post-OSKM transduction;

FIG. 13 shows a non-limiting example apoptosis and cell cycle profile ofDot1L-inhibited cells;

FIG. 14 shows a non-limiting example of the kinetics of iPSC colonyformation upon Dot1L knockdown;

FIG. 15 shows a non-limiting example of increased reprogrammingefficiency using small molecule inhibitor of Dot1L;

FIG. 16 shows a non-limiting example of reprogramming of Dot1Lconditional knockout tail-tip fibroblasts TTFs;

FIG. 17 shows a non-limiting example of knockdown efficiency of Nanogand Lin28 during 2-factor reprogramming;

FIG. 18 illustrates a non-limiting example of Chip-seq experimentaldesign;

FIG. 19 shows a non-limiting example of the relationship betweenH3K79me2 and H3K27me3; and,

FIG. 20 shows a non-limiting example of genes marked with K79me2specifically in fibroblasts, in ESCs and in both cell types.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the disclosure provides methods and compositions that areuseful in the production of stem cells (e.g., induced pluripotent stemcells—iPSC) from differentiated cells. In some embodiments, theinhibition of Dot1L, SUV39H1, and/or YY1 during at least a portion of acell reprogramming technique increases the efficiency of and/oraccelerates the reprogramming process. In some embodiments, theinhibition of one or more of these proteins also can be used to reducethe number of transcription factors required for a cell reprogrammingprocedure.

Stem cells produced using compositions and methods described herein areuseful in therapeutic and/or research applications. In addition, methodsand compositions of promoting stems cell production can be used fortherapeutic and/or research purposes.

Methods and compositions described herein can simplify the process ofreprogramming a somatic cell. In general, the production of inducedpluripotent stem cells (iPSCs) from differentiated cell types by somaticcell reprogramming involves resetting the epigenetic states of thedifferentiated cells. Typical reprogramming techniques involve exposingcells to several transcription factors, some of which can haveundesirable side-effects (e.g., they can be oncogenic). Surprisingly,inhibiting one or more of Dot1L, SUV39H1, and/or YY1 during at least aportion of a reprogramming technique allows for the use of fewertranscription factors, thereby reducing the risks associated withcertain transcription factors, in addition to enhancing the efficiencyand speed of the overall reprogramming procedure.

In some aspects, the disclosure also provides compositions and methodsfor identifying genes and proteins that are negative or positiveregulators in the reprogramming of differentiated cells. While severalproteins are known to regulate chromatin marks associated with thedistinct epigenetic states of cells before and after reprogramming, howchromatin-modifying proteins influence the reprogramming process remainslargely unknown. By identifying negative or positive regulators of iPSCgeneration, techniques for controlling the process of iPSC generationcan be further developed or refined by inhibiting or increasing theexpression and/or activity of one or more of the negative or positiveregulators.

In some embodiments, inhibition of the core components of the polycombrepressive complex 1 and 2, including the histone 3 lysine 27methyltransferase Ezh2, reduced reprogramming efficiency. However,surprisingly, inhibition of SUV39H1, YY1, and Dot1L, increasedreprogramming. In contrast to genes whose functions appear to berequired for reprogramming, inhibition of these three genes enhancedreprogramming (see FIG. 1D). YY1 is a transcription factor thatactivates or represses transcription in a context-dependentmanner^(10,11), whereas Suv39H1 is a histone H3K9 methyltransferaseimplicated in heterochromatin formation¹², and Dot1L is a H3K79 histonemethyl-transferase.

In some embodiments, inhibition of Dot1L, the H3K79 histonemethyl-transferase, either by RNAi or by a small molecule inhibitoraccelerated reprogramming, significantly increased the yield of iPSCcolonies, and substituted for Klf4 and c-Myc in a reprogrammingcocktail. In some embodiments, inhibition of Dot1L functions early inthe reprogramming process can be used to markedly induce two alternativereprogramming factors, Nanog and Lin28. Furthermore, in loss-of-functionexperiments, it was shown that Nanog and Lin28 play essential functionalroles in the enhancement of reprogramming by Dot1L-inhibition in someembodiments.

As shown herein, suppression of Dot1L expression using shRNAs orinhibition of its catalytic activity using a small molecule bothaccelerates and increases the yield of iPSCs and substitutes for bothKlf4 and Myc in the reprogramming process. These effects are primarilymediated through induction of two key pluripotency factors, Nanog andLin28, whose activation normally occurs during the later stages ofreprogramming^(20,21,22,23). Accordingly, in some embodiments, cellreprogramming can be promoted (e.g., ex vivo) by inhibiting Dot1L and/orstimulating Nanog and/or Lin28 expression and/or activity.

Genome-wide analysis of K79me2 distribution revealed thatfibroblast-specific, epithelial to mesenchymal transition-associatedgenes start to lose K79me2 in the initial phases of reprogramming andDot1L inhibition facilitates the loss of this mark from such genes thateventually get repressed in the pluripotent state. Accordingly, in someembodiments these marks can be used to evaluate and/or monitor theeffectiveness of Dot1L inhibition in a cell reprogramming procedure thatis used to generate induced pluripotent stems cells.

It should be appreciated that in some embodiments, stem cells can begenerated from cultured somatic cell lines. However, in someembodiments, methods and compositions described herein can be used togenerate “personalized” stem cells (e.g., for autologous cell therapy)by obtaining one or more somatic cells from a subject and reprogrammingthe cell(s) ex vivo as described herein. These personalized stem cellscan be useful to produce cell preparations (e.g., containingundifferentiated stem cells and/or differentiated stem cells) that canbe reintroduced to the subject (e.g., to treat a disease or disorder)with a low risk of host rejection of the reimplanted cells.

In some embodiments, inhibition of Dot1L can be used to prepare cellsfor a reprogramming procedure by inhibiting Dot1L prior to exposing thecells to a reprogramming cocktail (e.g., containing one or moretranscription factors that are useful to promote reprogramming).However, in some embodiments, Dot1L inhibition can be initiated duringreprogramming. In some embodiments, one or more Dot1L inhibitors areprovided along with one or more transcription factors in a reprogrammingcocktail. In some embodiments, one or more Dot1L inhibitors are added toa reprogramming cocktail that contains one or more transcriptionfactors. Accordingly, it should be appreciated that cells beingreprogrammed can be contacted with (e.g., incubated with) one or moreDot1L inhibitors (and/or one or more YY1 inhibitors and/or one or moreSUV39H1 inhibitors) prior to, along with, and/or after incubation withone or more transcription factors in an incubation cocktail. Thepresence of the inhibitor(s) can be useful to increase the efficiency ofthe reprogramming, accelerate the reprogramming (e.g., reduce theincubation time with the reprogramming cocktail), and/or reduce thenumber of transcription factors required in the incubation cocktail.

Stem cells described herein can be used for therapeutic and researchapplications. In some embodiments, stem cells can be administered to asubject (e.g., a human subject). In some embodiments, stem cells can bedifferentiated into one or more somatic cell types of interest prior toadministration to a subject. In some embodiments, stem cells can be usedto produce artificial tissue or organs ex vivo (e.g., for organtransplantation purposes). Techniques for differentiating stem cellsinto different types of cell types (e.g., prior to administration to asubject or for use in generating artificial tissue or organs) are knownin the art.

Although a cell reprogramming technique typically occurs ex vivo usingisolated cells that are obtained from a cell bank or a subject, methodsand compositions described herein also can be used in vivo to promotestem cell production and or maintenance in a subject. For example, insome embodiments one or more inhibitors described herein can beadministered to a subject (e.g., using any suitable route) to promote ormaintain stem cell populations in the subject. In some embodiments, theinhibitor(s) may be administered systemically. In some embodiments, theinhibitor(s) may be administered locally (e.g., to provide localstimulation or maintenance of a stem cell population). In someembodiments, one or more inhibitors may be administered in combinationwith one or more other factors, including but not limited to, one ormore transcription factors (e.g., 2, 3, 4 or more transcription factorssuch as those described herein). In some embodiments, the transcriptionfactors are provided in the form of a transcription factor expressingvector that expresses one or more transcription factors in vivo afteradministration. In some embodiments, a vector can include one or moreexpression regulators to limit expression of the transcription factor(s)to particular tissue or cell types. In some embodiments, theinhibitor(s) and transcription factor(s) are administered locally.Accordingly, methods and compositions described herein can be used toinduce local stem cell populations.

In some embodiments, one or more small molecule inhibitors of Dot1L, YY1and/or SUV39H1 are provided to cells (e.g., ex vivo, or in vivo) in anamount sufficient to inhibit protein function. In some embodiments, thecells are exposed to 0.01 uM, 0.1 uM, 1 uM or 10 uM of inhibitor for atime sufficient to inhibit the protein during a reprogramming procedure.In some embodiments, the cells are exposed to the inhibitor for severalhours or several days (e.g., 1-10 days, for example about 5 days)before, during, or after incubation with a reprogramming cocktail.

In some embodiments, one or more of the inhibitors described herein (forexample one or more inhibitors of Formula I, II, III, or IV) may be usedto inhibit Dot1L in vivo or in vitro. In some embodiments, an inhibitorof structure:

pharmaceutically acceptable salt thereof

may be used to inhibit Dot1L in vitro (e.g., contacted to differentiatedcells in vitro, for example alone or in combination with one or moretranscription factors or other agents described herein, to promote iPSCgeneration in vitro) or in vivo (e.g., administered to a subject invivo, for example alone or in combination with one or more transcriptionfactors or other agents described herein, to promote iPSC generation invivo, for example to promote local stem cell production or to supportlocal stem cell growth or maintenance).

In some embodiments, Dot1L, YY1 and/or SUV39H1 is inhibited bycontacting a differentiated cell with one or more nucleic acids thatprevent production of the protein(s). In some embodiments, Dot1L, YY1and/or SUV39H1 is inhibited by an shRNA that knocks down expression.Non-limiting embodiments of shRNAs that knock down expression areprovided in the Examples section.

Nucleic acids that prevent Dot1L, YY1 and/or SUV39H1 expression can beprovided to the cells in a variety of formats including dsRNA, siRNA andshRNA. “RNA interference (RNAi)” is an evolutionally conserved processwhereby the expression or introduction of RNA of a sequence that isidentical or highly similar to a target gene results in the sequencespecific degradation or specific post-transcriptional gene silencing(PTGS) of messenger RNA (mRNA) transcribed from that targeted genethereby inhibiting expression of the target gene. In one embodiment, theRNA is double stranded RNA (dsRNA). This process has been described inplants, invertebrates, and mammalian cells. In nature, RNAi is initiatedby the dsRNA-specific endonuclease Dicer, which promotes processivecleavage of long dsRNA into double-stranded fragments termed siRNAs.“Short interfering RNA” (siRNA), also referred to herein as “smallinterfering RNA” is defined as a nucleic acid-comprising agent whichfunctions to inhibit expression of a target gene, by RNAi. An siRNA maybe chemically synthesized, may be produced by in vitro transcription, ormay be produced within a host cell. In one embodiment, siRNA is a doublestranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides inlength, preferably about 15 to about 28 nucleotides, more preferablyabout 19 to about 25 nucleotides in length, and more preferably about19, 20, 21, 22, or 23 nucleotides in length, and may contain a 3′ and/or5′ overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the overhang on one strand is not dependenton the length of the overhang on the second strand. Preferably the siRNAis capable of promoting RNA interference through degradation or specificpost-transcriptional gene silencing (PTGS) of the target messenger RNA(mRNA). siRNAs also include small hairpin (also called stem loop) RNAs(shRNAs). In one embodiment, these shRNAs are composed of a short (e.g.,about 19 to about 25 nucleotide) antisense strand, followed by anucleotide loop of about 5 to about 9 nucleotides, and the analogoussense strand. Alternatively, the sense strand may precede the nucleotideloop structure and the antisense strand may follow. These shRNAs may beencoded by plasmids, retroviruses, and lentiviruses and expressed from,for example, the pol III U6 promoter, or another promoter (see, e.g.,Stewart, et al., RNA April; 9(4):493-501 (2003), incorporated byreference herein in its entirety).

It is envisioned that inhibitors of Dot1L, YY1 and/or SUV39H1 are usedin effective amounts. In some embodiments, an effective amount of Dot1Linhibitor (e.g., small molecule or RNAi molecule) is an amountsufficient to inhibit the transferase activity (e.g., by at least 25%,at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 99%, or more) in the targeted cells (e.g., cellsgrowing in vitro, or targeted cells types in a subject). In someembodiments, an effective amount of YY1 inhibitor is an amountsufficient to inhibit the activity (e.g., by at least 25%, at least 50%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99%, or more) in the targeted cells (e.g., cells growing in vitro,or targeted cells types in a subject). In some embodiments, an effectiveamount of SUV39H1 inhibitor is an amount sufficient to inhibit theactivity (e.g., by at least 25%, at least 50%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 99%, or more) inthe targeted cells (e.g., cells growing in vitro, or targeted cellstypes in a subject).

In some embodiments, a subject may be evaluated to determine whetherDot1L is overexpressed. Certain genetic abnormalities associated withDot1L (e.g., the Dot1L regulatory pathway) result in Dot1Loverexpression. Assays for detecting genetic abnormalities associatedwith Dot1L overexpression are known in the art and can include, but arenot limited to, molecular assays (e.g., hybridization or sequencing) orin situ or chromosomal assays (e.g., FISH) or other chromosomal assays.Depending on the level of Dot1L in the subject (or in cells obtainedfrom the subject) the amount of Dot1L inhibitor that is used (e.g., invivo or in vitro) may be adjusted. One of ordinary skill can performassays to determine suitable levels of Dot1L inhibitor to use in orderto inhibit Dot1L levels that are present in a subject (for example invivo or in vitro in cells isolated from the subject).

Reprogramming

In some embodiments, inhibiting Dot1L, YY1 and/or SUV39H1 can be used topromote cellular reprogramming (e.g., ex vivo or in vivo). Duringdevelopment, individual cells become specified to adopt distinct fatesthrough the activation of lineage-specific gene expression programs.Under normal circumstances, these gene expression programs are stablyinherited throughout mitotic divisions, thereby maintaining cellidentity. At the molecular level, this cellular identity is largelycontrolled at the chromatin level whereby genes appropriate for a givenlineage are embedded in a chromatin structure that supports theirtranscriptional activity, whereas genes specifying other lineages aresequestered in repressive chromatin structures resulting intranscriptional silencing¹. Differentiated cells such as fibroblasts canbe converted into induced pluripotent stem cells (iPSCs) uponoverexpression of critical transcriptional regulators of embryonic stemcells (ESCs)^(2,3,4).

In some embodiments, the effect of inhibition of one or more proteins onreprogramming was evaluated in dH1fs cells. The dH1fs cells weretransfected with shRNA pools (at high multiplicity of infection toensure all cells received an shRNA vector) followed by super-infectionwith reprogramming vectors expressing Oct4, Sox2, Klf4 and c-Myc (OSKM),and the resulting iPSCs were identified by Tra-1-60 staining.

In one aspect, the disclosure provides a method of producing inducedpluripotent stem cells, by inhibiting Dot1L (and/or YY1 and/or SUV39H1)in a differentiated cell and culturing the differentiated cell underreprogramming conditions to produce induced pluripotent stem cells. Insome embodiments, the reprogramming conditions comprise the presence ofa reprogramming cocktail. In some embodiments, the reprogrammingcocktail comprises Oct4 and/or Sox2. In some embodiments, thereprogramming cocktail includes Klf4 and/or c-Myc. In some embodiments,the reprogramming cocktail does not include Klf4 and/or c-Myc. In someembodiments, the reprogramming cocktail consists essentially of Oct4 andSox2. In some embodiments, Dot1L (and/or YY1 and/or SUV39H1) isinhibited at the same time as providing the reprogramming cocktail.

Reprogramming, when relating to cells, generally refers to the processof changing a cell from a first phenotype to a second phenotype. Inparticular, and as used herein, reprogramming refers to the process ofchanging a differentiated cell into an induced pluripotent stem cell.

Reprogramming conditions as used herein refers to the conditions thatallow a differentiated cell to transform into an induced pluripotentstem cell. Reprogramming conditions include culture media, culturecomponents (e.g., buffer, pH, salt) and the duration of the culturing ofthe cells.

In some embodiments, the reprogramming conditions comprise the presenceof a reprogramming cocktail. A reprogramming cocktail as used hereinrefers to the combination of specific agents (e.g., Sox2, Oct 4, Klf4,c-Myc, or a combination of any 2 or 3 thereof, or all 4, and/or one ormore additional transcription factors) that is required to reprogram acell from a differentiated cell into an induced pluripotent stem cell.In some embodiments, the reprogramming cocktail comprises Oct4 and Sox2.In some embodiments, the reprogramming cocktail consists essentially ofOct4 and Sox2. A reprogramming cocktail that consists essentially ofOct4 and Sox2, refers to a reprogramming cocktail that does not includeany other specific agents (e.g., transcription factors) that could beused to reprogram a differentiated cell. However, in some embodiments, areprogramming cocktail (e.g., a cocktail that consists essentially ofspecified transcription factors) may include buffers, salts, sugars, andother components that may be useful to support the growth andreprogramming of the differentiated cells.

The production of induced pluripotent stem cells is generally achievedby the introduction of nucleic acid sequences encoding stemcell-associated genes into a somatic cell. In general, these nucleicacids are introduced using retroviral vectors and expression of the geneproducts results in cells that are morphologically and biochemicallysimilar to pluripotent stem cells (e.g., embryonic stem cells). Thisprocess of altering a cell phenotype from a somatic cell or progenitorcell phenotype to a stem cell-like phenotype is termed “reprogramming”.

It was surprisingly found herein that inhibiting DotL1 provides analternative route for reprogramming cell, eliminating the need for oneor more cell transforming factors (e.g., c-Myc) that are associated withoncogenesis.

In some embodiments, reprogramming can be achieved by introducing acombination of stem cell-associated genes including, for example Oct3/4(Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2, Klf4,Klf5, c-Myc, 1-Myc, n-Myc and LIN28. In general, successfulreprogramming is accomplished by introducing Oct-3/4, a member of theSox family, a member of the Klf family, and a member of the Myc familyto a somatic or progenitor cell. In some embodiments, the nucleic acidsequences of Oct-4, Sox2, c-MYC, and Klf4 are delivered using a viralvector, such as an adenoviral vector, a lentiviral vector or aretroviral vector. However, while it is understood that reprogramming isusually accomplished by viral delivery of stem-cell associated genes, itis also contemplated herein that reprogramming can be induced usingother delivery methods.

In one aspect, the disclosure provides a method of accelerating theproduction of induced pluripotent stem cells, the method comprisinginhibiting Dot1L (and/or YY1 and/or SUV39H1) in a differentiated celland culturing the differentiated cell under reprogramming conditions toaccelerate the production of induced pluripotent stem cells, wherein theproduction of induced pluripotent stem cells is accelerated compared toa differentiated cell in which Dot1L is not inhibited. Thus, as providedherein, in some embodiments, a differentiated cell can be reprogrammedinto an induced pluripotent stem cells by using a known combination ofcell-associated genes (e.g., Oct-4, Sox2, c-MYC, and Klf4 or a subsetthereof). It was unexpectedly shown herein that the addition of Dot1L tothe combination of cell-associated genes resulted in the acceleration ofthe production of induced pluripotent stem cells.

The efficiency of reprogramming (e.g., the number of reprogrammed cells)can be enhanced by the addition of various small molecules as shown byShi, Y., et al (2008) Cell-Stem Cell. 2:525-528, Huangfu, D., et al(2008) Nature Biotechnology 26(7):795-797, Marson, A., et al (2008)Cell-Stem Cell 3:132-135, which are incorporated herein by reference intheir entirety. It is contemplated that the methods described herein canalso be used in combination with a single small molecule (or acombination of small molecules) that enhances the efficiency of inducedpluripotent stem cell production. Some non-limiting examples of agentsthat enhance reprogramming efficiency include soluble Wnt, Wntconditioned media, BIX-01294 (a G9a histone methyltransferase),PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histonedeacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine,dexamethasone, suberoylanilide, hydroxamic acid (SAHA), trichostatin(TSA), and inhibitors of the TGF-β signaling pathway, among others. Itis also contemplated herein that inhibitors can be used alone or incombination with other small molecule(s) to replace one or more of thereprogramming factors used for the production of induced pluripotentstem cells.

In some embodiments, a method of producing pluripotent stem cellsincludes upregulating the expression of Nanog and/or Lin28 in adifferentiated cell and culturing the differentiated cell underreprogramming conditions to produce induced pluripotent stem cells. Insome embodiments, Nanog and/or Lin28 are upregulated by inhibitingDot1L. In some embodiments, Nanog and/or Lin28 are upregulated byintroducing agents that stimulate expression of Nanog and/or Lin28,and/or by introducing agents that suppress removal of Nanog and/or Lin28from the cell (e.g., by protease action). In some embodiments, promotersare introduced into the genome of the cell that result in theupregulation of the expression of Nanog and/or Lin28.

In some embodiments, a method of producing pluripotent stem cellsincludes inhibiting SUV39H1 in a differentiated cell and culturing thedifferentiated cell under reprogramming conditions to produce inducedpluripotent stem cells. Histone-lysine N-methyltransferase SUV39H1 is amember of the suppressor of variegation 3-9 homolog family and encodes aprotein with a chromodomain and a C-terminal SET domain. This nuclearprotein moves to the centromeres during mitosis and functions as ahistone methyltransferase, methylating Lys-9 of histone H3Histone-lysine N-methyltransferase SUV39H1. It was surprisingly shownherein that inhibiting SUV39H1 provides a novel pathway for producinginduced pluripotent stem cells. In some embodiments, SUV39H1 isinhibited by providing the cell with an siRNA, shRNA (or other RNAi)against SUV39H1. In some embodiments, SUV39H1 is inhibited by providingthe cell with an SUV39H1 inhibitor (e.g., a small molecule inhibitorsuch as Chaetocin, see Greiner et al. Nat Chem Biol. 2005 August;1(3):143-5).

In some embodiments, a method of producing pluripotent stem cellsincludes inhibiting YY1 in a differentiated cell and culturing thedifferentiated cell under reprogramming conditions to produce inducedpluripotent stem cells. Transcriptional repressor protein YY1 is aubiquitously distributed transcription factor belonging to theGLI-Kruppel class of zinc finger proteins. The protein is involved inrepressing and activating a diverse number of promoters. YY1 may directhistone deacetylases and histone acetyltransferases to a promoter inorder to activate or repress the promoter, thus implicating histonemodification in the function of YY1. It was surprisingly shown hereinthat inhibiting YY1 provides a novel pathway for producing inducedpluripotent stem cells. In some embodiments, YY1 is inhibited byproviding the cell with an siRNA, shRNA (or other RNAi) against YY1. Insome embodiments, YY1 is inhibited by providing the cell with an YY1inhibitor (e.g., a small molecule inhibitor).

Differentiated Cells

Methods described herein embrace the use of any differentiated cell(e.g., somatic cell). In some embodiments, the differentiated cell is afibroblast, epithelial, endothelial, neuronal, adipose, cardiac,skeletal muscle, immune cells, hepatic, splenic, lung, circulatingblood, gastrointestinal, renal, bone marrow, or pancreatic cell. Thedifferentiated cell can be a primary cell isolated from any somatictissue including, but not limited to brain, liver, lung, gut, stomach,intestine, fat, muscle, uterus, skin, spleen, endocrine organ, bone,etc., Methods for isolating differentiated cells from a body are knownin the art.

In some embodiments, the differentiated cell is a fibroblast. In someembodiments, the differentiated cell is an adult fibroblast.

The differentiated cell used according to the methods provided hereincan be from any mammalian species, with non-limiting examples includinga murine, bovine, simian, porcine, equine, ovine, or human cell.

In some embodiments, the differentiated cell is a human cell. In someembodiments, the differentiated cell is dH1fs, IMR-90 or MRC-5.

In some embodiments, the differentiated cell is a mouse cell.

The differentiated cells used in the methods provided herein, and priorto being reprogrammed, can be maintained under in vitro conditions usingconventional tissue culture.

In some embodiments, one or more differentiated cells are obtained froma subject that will subsequently be treated by reimplanting inducedpluripotent stem cells obtained from the differentiated cells and/or byreimplanting redifferentiated cells, tissue, or organs obtained from thestem cells.

Induced Pluripotent Stem Cells

In one aspect, the disclosure provides methods for producing stem cells.In some embodiments, the cells are induced pluripotent stem cells. Theterm “pluripotent,” as used in the context of cells, describes thedevelopmental capacity of a cell or cell population and refers to cellsthat are capable of self-renewal and have the capacity to differentiateinto cells of any of the three germ layers (endoderm, mesoderm, andectoderm). Examples of pluripotent cells are embryonic stem cells(ES-cells), embryonic carcinoma cells (EC cells), and inducedpluripotent stem cells (iPS cells). A pluripotent cell has the potentialto give rise to any fetal or adult cell type. However, pluripotent cellscannot contribute to extraembryonic tissue, such as, the placenta. Thisdistinguishes pluripotent cells from totipotent cells, which can giverise to all fetal, adult, and extraembryonic cell types.

In one aspect of the methods provided herein, the presence of inducedpluripotent stem cells is determined. In some embodiments, the presenceof induced pluripotent stem cells is determined by evaluating thepresence of one or more markers selected from the group consisting ofSSEA4, SSEA3, Tra-1-81, Oct4, Sox2 and Nanog.

In humans and mice; the transcription factors Oct-4, Sox2, and Nanog arebiomarkers that are specific for pluripotent cells (see, e.g.,Looijenga, L. H. et al. (2003) Cancer Res 63, 2244-50; Peske, M. andSchöler, H. R. (2001) Stem Cells 19, 271-8; Pan, G. and Thomson, J. A.(2007) Cell Res 17, 42-9; the entire contents of each of which areincorporated herein by reference). Additional biomarkers specific forhuman pluripotent cells include stage specific embryonic antigens SSEA3and SSEA4, as well as the podocalyxin antigens TRA 1-81 and TRA 1-60(see, e.g., Schopperle et al. (2007) Stem Cells 25(3):723-30. Epub 2006Nov. 22; Henderson, J. K. et al. (2002) Stem Cells 20, 329-37; andDraper, J. S. et al. (2002) J Anat 200, 249-58; the entire contents ofeach of which are incorporated herein by reference). Additionalbiomarkers for mouse pluripotent stem cells include SSEA1, which isspecific for pluripotent cells, and the less stringent marker alkalinephosphatase, which is indicative, but not specific for pluripotent cells(see, e.g., Brambrink, T. et al. (2008) Cell Stem Cell 2(2):151-9; theentire contents of which are incorporated herein by reference).Expression of these pluripotency markers decreases and is eventuallylost during differentiation, corresponding to a restriction indevelopmental potential and, thus, a loss of pluripotency (see, e.g.,Schopperle, W. M. and DeWolf, W. C. (2007) Stem Cells 25, 723-30; theentire contents of which are incorporated herein by reference). Table 1below lists some additional biomarkers useful for the identification ofpluripotent stem cells from mouse, human, and other mammals, as well astheir expression spectrum among pluripotent cell types.

TABLE 1 stem cell markers Biomarker Expressed in Remarks Alkaline ES, ECElevated expression of this enzyme is associated phosphatase withundifferentiated pluripotent stem cell (PSC) Cluster ES, EC Surfacereceptor molecule found specifically on designation 30 PSC (CD30) Cripto(TDGF-1) ES, Growth factor expressed by ES cells, primitivecardiomyocyte ectoderm, and developing cardiomyocyte GCTM-2 ES, ECExtracellular-matrix antigen that is synthesized by undifferentiatedPSCs Genesis ES, EC Transcription factor expressed by PSCs Germ cellnuclear ES, EC Transcription factor expressed by PSCs factor OCT4/POU5F1ES, EC Transcription factor unique to PSCs Stage-specific ES, ECGlycoprotein specifically expressed in early embryonic embryonicdevelopment and by undifferentiated PSCs antigen-3 (SSEA-3)Stage-specific ES, EC Glycoprotein specifically expressed in earlyembryonic embryonic development and by undifferentiated PSCs antigen-4(SSEA-4) Stem cell factor ES, EC, HSC, Membrane protein that enhancesproliferation of ES (SCF or c-Kit MSC and EC cells, hematopoietic stemcell (HSCs), and ligand) mesenchymal stem cells (MSCs); binds thereceptor c-Kit Telomerase ES, EC An enzyme uniquely associated withimmortal cell lines; useful for identifying undifferentiated PSCsTRA-1-60 ES, EC Antibody to a specific extracellular matrix molecule issynthesized by undifferentiated PSCs TRA-1-81 ES, EC Antibody to aspecific extracellular matrix molecule normally synthesized byundifferentiated PSCs

In some embodiments, methods provided herein include a step of isolatingthe produced induced pluripotent stem cells. In some embodiments, theinduced pluripotent stem cells are isolated from a mixed populationcells comprising pluripotent cells and cells that have not yet or onlypartially been reprogrammed. Methods for isolating induced pluripotentstem cells are known in the art and are generally based on moieties thatbind to markers that are uniquely expressed on induced pluripotent stemcells (e.g., SSEA-1).

In some embodiments, methods described herein include a step ofevaluating the developmental capacity of the induced pluripotent stemcells. In some embodiments, the compositions of induced pluripotent stemcells provided herein can be evaluated for their developmental capacity.In some embodiments, the induced pluripotent stem cells are evaluatedfor their capacity to differentiate into ectoderm, mesoderm and endodermcells. In some embodiments, the developmental capacity of the inducedpluripotent stem cells is evaluated in a teratoma assay.

The developmental capacity of a cell or a cell population can be testedby assays well known to those of skill in the art. One test forpluripotency of a mouse cell or a population of mouse cells, is diploidblastocyst complementation, in which one or more of the cells inquestion are injected into a host blastocyst, which is then transferredto a foster mouse. If the injected cell(s) are pluripotent, contributionto all three germ layers, and to the germ line, will be observed in theresulting pup, which will typically be a chimera comprising cellsderived from the host blastocyst and from the injected cell(s). The moststringent test for the developmental capacity of a pluripotent mousecell, e.g., an ES or iPS cell is the tetraploid blastocystcomplementation assay, which is well known to those of skill in the art(see, e.g., Eggan et al., PNAS, 2001 (May) 6209-6214; and Li et al.,Reproduction, 2005 130:53-59; the entire contents of which areincorporated herein by reference). In this assay, a cell or a pluralityof cells is injected into a tetraploid host blastocyst. The cells of thetetraploid host blastocyst can contribute to extra-embryonic tissues,e.g., the placenta, but cannot give rise to fetal or adult cell types.Accordingly, a tetraploid blastocyst alone cannot give rise to a livepup at birth, because it cannot generate the required fetal tissues.However, if a pluripotent cell or a plurality of such cells is injectedinto a tetraploid blastocyst, thus creating a complemented tetraploidblastocyst, the pluripotent cell(s) can give rise to the cell typesrequired for embryonic development, if the injected cells exhibit a highdevelopmental capacity. Tetraploid blastocyst complementation is a morestringent test for developmental capacity than the derivation ofchimeric mice after diploid blastocyst injection, because any defect indevelopmental capacity of the injected cell(s) cannot be compensated bycells of the host blastocyst. A pup resulting from a complementedtetraploid blastocyst typically consists of cells derived from theinjected cell(s).

Additional tests for developmental capacity or pluripotency includeteratoma formation assays, also sometimes referred to as teratocarcinomaassays (see. e.g., Wesselschmidt, R. L. (2011) Methods Mol Biol.767:231-41, the entire contents of which are incorporated herein byreference). Typically, a population of cells to be tested is injectedsubcutaneously into an immunocompromised host animal, e.g., a SCIDmouse. If the cell population comprises pluripotent cells, a solid tumorwill form at the site of injection. Teratocarcinomas derived frompluripotent cells will contain differentiated cell types of all threegerm layers. This assay is an in vivo assay for differentiation capacitythat can be used for all pluripotent cells, including cells that cannotbe tested by blastocyst complementation (e.g., human cells).

In some embodiments, methods described herein include a step ofpromoting the development and/or differentiation one or more of theinduced pluripotent stem cells.

In some embodiments, the iPSCs produced as described herein may bedifferentiated in vitro, partially or completely. Methods of promotingthe development or differentiation of stem cells are known in the art.For example, differentiation protocols are known in the art and includethose described in U.S. Pat. No. 7,326,572 (endoderm differentiation),U.S. Pat. No. 7,282,366 (hepatocyte differentiation), U.S. Pat. No.7,250,294 (neural differentiation), and U.S. Pat. No. 7,033,831 (isletcell differentiation). In some embodiments, Embryoid Bodydifferentiation, or in vitro directed differentiation using cytokines,growth factors, and/or co-culture with mature cell types may be used. Avariety of differentiation factors that can act on pluripotent stemcells and their precursor progeny are known in the art. For example,members of the BMP family of factors have been used to differentiatepluripotent stem cells such. These include the use of BMP-4 and BMP-7 togenerate endoderm-like differentiation. (Xu et al. Nat Biotechnol20:1261-1264, 2002; Pera et al. J Cell Sci 117:1269-1280, 2004.) ActivinA can be used to differentiate pluripotent stem cells into definitiveendoderm using monolayers or three dimensional (e.g., EB) culturesystems. (D'Amour et al. Nat Biotechnol 23:1534-1541, 2005.) Nervoussystem cells have been observed as a result of culture with epidermalgrowth factor and fibroblast growth factor (resulting in the generationof neurospheres that comprise neural stem cells), subsequent removal ofthese factors (resulting in the generation of astrocyte-like cells) orsupplementation with nerve growth factor (resulting in the generation ofneurons and glial cells). (Kim et al. Nature 418:50-6, 2002; Lee et al.Nat Biotechnol 18:675-9, 2000.) Dopaminergic neurons, useful inParkinson's disease, may be formed through culture or contact with FGF20and FGF2. Bjorklund et al. (PNAS 2002, 99:2344-2349) provides additionalmethods for differentiating ES cells into dopaminergic neurons. Hepaticcell differentiation may be induced through contact and/or culture withan insulin, dexamethasone, and collagen type I (via EB formation)combination; a sodium butyrate and DMSO combination; an FGF4, HGF andcollagen type I combination; an aFGF, HGF, oncostatin M, dexamethasoneand collagen type I combination; and a bFGF, variant HGF, DMSO anddexamethasone combination in the presence of poly-amino-urethane coatednon-woven polytetrafluoroethylene fabric. (Shirahashi et al. CellTransplant 13:197-211, 2004; Rambhatla et al. Cell Transplant 12:1-11,2003; Schwartz et al. Stem Cells Dev 14:643-655, 2005; Baharvand et al.Int J Dev Biol 50:645-652, 2006; Soto-Gutierrez et al. Cell Transplant15:335-341, 2006.) Hepatic differentiation may also occur spontaneously.(Lavon et al. Differentiation 72:230-238, 2004.) Pancreaticdifferentiation, including differentiation towards beta-islet cells, canbe induced using Activin A, retinoic acid, FGF2 and FGFIO, betacellulin,HGF, Exendin 4, DKK1 and DKK3. (Gu et al. Mech Dev 120:35-43, 2003;Grapin-Botton et al. Trends Genet 16:124-130, 2000; D'Amour et al. NatBiotechnol 23:1534-1541, 2005a; D'Amour et al. published US applicationUS2005-0266554A1.) Endothelial differentiation may be induced in thepresence of ECM proteins such as collagen type IV, optionally in thepresence of VEGF and bFGF. (Gerecht-Nir et al. Lab Invest 83:1811-1820,2003.) Further reference may be made to published PCT applicationWO2009/007852 for a review of various differentiative procedures knownin the art and applicable to the differentiation of the immature andprecursor cells of the invention. Such teachings, and in particularthose found on pages 57-61 (under the subheading “Cell Differentiation”)of WO2009/007852, are incorporated by reference herein. Still otherreferences include West and Daley, 2004, Curr Opin Cell Biol 16:688-692;U.S. Pat. No. 6,534,052 B1; Kehat and Gepstein, 2003, 8:229-236; Nir etal., 2003, 58:313-323; and U.S. Pat. Nos. 6,613,568 and 6,833,269.

In some embodiments, the disclosure provides compositions comprising apopulation of induced pluripotent stem cells (e.g., human inducedpluripotent stem cells) produced according to the methods providedherein. In some embodiments, the disclosure provides compositionscomprising a population of differentiated stem cells (e.g., humandifferentiated stem cells) obtained from stem cells produced accordingto the methods provided herein. In some embodiments, a composition, inaddition to the population of induced pluripotent stem cells and/ordifferentiated stem cells, may include cell culture and cell culturecomponents needed for cell viability. In some embodiments, thecompositions include pharmaceutical excipients allowing for theadministration of the induced pluripotent stem cells.

It should be appreciated that differentiated cells (e.g., obtained froma subject), induced pluripotent stem cells, and/or differentiated iPSCsreferred to herein may be isolated cells (e.g., in the form ofpreparations of isolated cells). As used herein, isolated cells arecells which have been physically separated from their environment. Ifthe cells are naturally occurring, then isolation implies that the cellsare physically separated from the naturally occurring environment fromwhich they derive. In some instances, isolated cells are additionally oralternatively physically separated, in whole or in part, from an invitro environment. Thus, as used herein, the term isolated means that amolecule, cell, cell population and the like is physically separatedfrom an environment in which it normally exists, or in which itoriginally or previously existed. Isolation may refer to physicalseparation of cells from a culture condition (e.g., a differentiationculture), from a naturally occurring environment or source, and thelike. A differentiated cell population may be isolated from adifferentiation culture condition, for example, by harvesting the cellsand removing the culture medium (e.g., by centrifugation). Isolating mayalso involve washing the cells. Typically, the cells are resuspended infresh medium. Isolation of the differentiated cell population from thedifferentiation culture therefore can serve to remove factors or stimuliused to differentiated the pluripotent stem cells towards one or morelineages.

In some embodiments, cells described herein (e.g., originaldifferentiated cells, iPSCs, and or subsequently differentiated cells)may be genetically manipulated (e.g., they may be transfected) or theymay not be genetically manipulated. Transfection refers to geneticmanipulation of cells to introduce and typically express an exogenousnucleic acid. The exogenous nucleic acid may be a reporter such as greenfluorescent protein (GFP) or it may be a selection marker such asthymidine kinase or it may be a transcription factor or other factorthat is used to promote reprogramming or subsequent redifferentiation.It will be understood that in some instances reporters such as GFP mayalso serve as selection markers, particularly if their expression iscontrolled by pluripotent gene promoters such as an Oct4 promoter.

It should be appreciated that techniques described herein may be usedwith cells obtained from different species, including for example,human, and various animal species including household species such asdogs and cats, agricultural species such as cows, pigs, and horses,laboratory species such as mice and rats, and the like, or otherspecies.

In Vivo Uses

In some embodiments, the disclosure provides methods of treatment usingthe induced pluripotent stem cells or differentiated cells (e.g.,obtained from iPSCs) provided herein. In some embodiments, the methodsof treatment comprise administering to a person in need thereof acomposition comprising induced pluripotent stem cells and/ordifferentiated stems cells produced according to the methods providedherein.

In some embodiments, a person in need of treatment with a compositioncomprising induced pluripotent stem cells or differentiated cells is aperson having brain damage, cancer, spinal cord injury, heart damage,baldness, deafness, diabetes, neuronal defects, blindness, amyotrophiclateral sclerosis, a genetic disorder, infertility, or unhealed wounds.In some embodiments, cells obtained as described herein also may be usedto treat hematological malignancies, immunodeficiencies, age relatedmacular degeneration, and other conditions. For example cell populations(e.g., differentiated cell populations) can be used in transplantsettings in the treatment or prevention of various conditions includingbut not limited to Parkinson's disease (dopaminergic neurons),Alzheimer's disease (neural precursors), Huntington's disease (GABAergicneurons), blood disorders such as leukemia, lymphoma myeloma and anemia(hematopoietic cells), side-effects of radiation e.g., in transplantpatients (hematopoietic precursors), cardiovascular disease, myocardialinfarction, ischemic cardiac tissue or heart-failure (partially- orfully-differentiated cardiomyocytes), muscular dystrophy (skeletalmuscle cells), liver cirrhosis or failure (hepatocytes), chronichepatitis (hepatocytes), diabetes including type I diabetes(insulin-producing cells such as islet cells), ischemic brain damage(neurons), spinal cord injury (glial progenitor cells and motorneurons), amyotrophic lateral sclerosis (ALS) (motor neurons),orthopedic tissue injury (osteoblasts), kidney disease (kidney cells),corneal scarring (corneal stem cells), cartilage damage (chondrocytes),bone damage (osteogenic cells including osteocytes), osteoarthritis(chondrocytes), myelination disorders such as Pelizaeus-Merzbacherdisease, multiple sclerosis, adenoleukodystrophies, neuritis andneuropathies (oligodendrocytes), and hair loss.

Pluripotent or differentiated cell populations may be provided aspharmaceutical compositions that are sterile and appropriate for in vivouse, optionally together with a pharmaceutically acceptable carrier. Asused herein, a pharmaceutically-acceptable carrier means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients. Pharmaceuticallyacceptable carriers include diluents, fillers, salts, buffers,stabilizers, solubilizers and other materials which are well-known inthe art. Such preparations may routinely contain salt, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. Cell populations may be formulated for local or systemicadministration including as part of an implant. Cells may be used aloneor together with another agent, whether active or inactive, includingbut not limited to a scaffold, a matrix, and the like. These cells mayfurther be included in a kit that additionally comprises at a minimuminstructions for use of the cells, and optionally comprises one or moreother agents whether active or inactive. The cells may be provided as afrozen aliquot of cells, a culture of cells, or a liquid suspension ofcells.

Cells may be administered in numbers effective to produce a desiredresult, including but not limited to a short-term or long-termtherapeutic result. Such result may include an improvement in orcomplete eradication of symptoms associated with a particular condition.The cell numbers to be administered will depend on a number of factorsincluding the weight and age of the subject, the type of condition beingtreated, the desired effect (e.g., short-term or long-term), and thelike. Some treatments therefore may require as few as 10³ cells, whileothers may require 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or more cells.

In Vitro Uses

Cell populations produced as described herein may be studied for geneexpression profiles and/or responses to various external stimuli inorder to understand differentiation or other processes more fully. Insome embodiments, disease-specific iPS cells may be used to modeldiseases (e.g., ALS or other diseases). In some embodiments, cells maybe used in methods for screening and/or identifying agents (e.g.,therapeutic agents such as inhibitors) being used or to be usedclinically. These assays may measure the therapeutic efficacy and/ortoxicity of the candidate agent, among other things. The readouts fromsuch in vitro assays are correlative of the in vivo toxicity or efficacysuch agents would exhibit in subjects. Thus, the effect of the agent onthe differentiated cells generated according to the invention in vitrois a form of surrogate marker or readout for how the agent will functionin vivo in a subject. The agents to be tested include those usedclinically as well as experimental agents. In some more commonembodiments, such testing will focus on the toxicity of agents includingdrugs in particular differentiated progeny. Accordingly, in theseassays, the readout would be cell death (or conversely cell viability).These in vitro assays may employ suspensions of pluripotent ordifferentiated cells, adherent populations of pluripotent ordifferentiated cells, or three dimensional structures comprised ofpluripotent or differentiated cells (e.g., in vitro organ tissues,matrices and architectures).

Administration of Cell Preparations

In one aspect, the disclosure provides methods for administering inducedpluripotent stem cells or differentiated cells produced according tomethods provided herein.

Cells can be administered to hosts by a variety of methods as discussedelsewhere herein. In certain embodiments the cells are administered byinjection, such as by intravenous injection. In some embodiments cellsare encapsulated for administration. In some embodiments the cells areadministered in situ. In some embodiments of the invention, cells areadministered in doses measured by the ratio of cells to body mass(weight). Alternatively, iPSCs can be administered in doses of a fixednumber of cells.

In some embodiments the purity of cells for administration to a subjectis about 100%. In other embodiments it is 95% to 100%. In someembodiments it is 85% to 95%. Particularly in the case of admixtureswith other cells, the percentage of IPSCs or differentiated cells can be25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%, 80%-90%,or 90%-95%.

The number of iPSCs or differentiated cells in a given volume can bedetermined by well known and routine procedures and instrumentation. Thepercentage of iPSCs or differentiated cells in a given volume of amixture of cells can be determined by much the same procedures. Cellscan be readily counted manually or by using an automatic cell counter.Specific cells can be determined in a given volume using specificstaining and visual examination and by automated methods using specificbinding reagent, typically antibodies, fluorescent tags, and afluorescence activated cell sorter.

The choice of formulation for administering cells for a givenapplication will depend on a variety of factors. Prominent among thesewill be the species of subject, the nature of the disorder, dysfunction,or disease being treated and its state and distribution in the subject,the nature of other therapies and agents that are being administered,the optimum route for administration of the cells, survivability ofcells via the route, the dosing regimen, and other factors that will beapparent to those skilled in the art. In particular, for instance, thechoice of suitable carriers and other additives will depend on the exactroute of administration and the nature of the particular dosage form,for example, liquid dosage form (e.g., whether the composition is to beformulated into a solution, a suspension, gel or another liquid form,such as a time release form or liquid-filled form).

Examples of compositions comprising iPSCs and/or differentiated cellsinclude liquid preparations, including suspensions and preparations forintramuscular or intravenous administration (e.g., injectableadministration), such as sterile suspensions or emulsions. Suchcompositions may comprise an admixture of cells with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose, dextrose, or the like. The compositions can also belyophilized. The compositions can contain auxiliary substances such aswetting or emulsifying agents, pH buffering agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE,”17th edition, 1985, incorporated herein by reference, may be consultedto prepare suitable preparations, without undue experimentation.

Typically, the compositions will be isotonic, for example, they willhave the same osmotic pressure as blood and lacrimal fluid when properlyprepared for administration. The desired isotonicity of the compositionsof this invention may be accomplished using sodium chloride, or otherpharmaceutically acceptable agents such as dextrose, boric acid, sodiumtartrate, propylene glycol, or other inorganic or organic solutes.Sodium chloride is preferred particularly for buffers containing sodiumions.

A pharmaceutically acceptable preservative or cell stabilizer can beemployed to increase the life of cellular compositions. If suchpreservatives are included, it is well within the purview of the skilledartisan to select compositions that will not affect the viability orefficacy of the cells.

Sterile injectable solutions can be prepared by incorporating the cellsutilized in practicing the present invention in the required amount ofthe appropriate solvent with various amounts of the other ingredients,as desired.

For any composition to be administered to an animal or human, and forany particular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD50 in a suitable animal model, e.g., rodent such as mouse or rat; and,the dosage of the composition(s), concentration of components therein,and timing of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure, and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

Order of administration, formulations, doses, frequency of dosing, androutes of administration of factors (such as the cytokines discussedherein) and IPSCs generally will vary with the disorder or disease beingtreated, its severity, the subject, other therapies that are beingadministered, the stage of the disorder or disease, and prognosticfactors, among others. General regimens that have been established forother treatments provide a framework for determining appropriate dosingin cell-mediated direct or adjunctive therapy. These, together with theadditional information provided herein, will enable the skilled artisanto determine appropriate administration procedures in accordance withembodiments of the invention, without undue experimentation.

It should be appreciated that iPSCs or differentiated cells describedherein can be administered to a subject by any of a variety of routesknown to those skilled in the art that may be used to administer cellsto a subject.

Among methods that may be used in this regard in embodiments of theinvention are methods for administering cells by a parenteral route.Parenteral routes of administration useful in various embodiments of theinvention include, among others, administration by intravenous,intraarterial, intracardiac, intraspinal, intrathecal, intraosseous,intraarticular, intrasynovial, intracutaneous, intradermal,subcutaneous, and/or intramuscular injection. In some embodimentsintravenous, intraarterial, intracutaneous, intradermal, subcutaneousand/or intramuscular injection are used. In some embodimentsintravenous, intraarterial, intracutaneous, subcutaneous, and/orintramuscular injection are used.

Cells may be administered to the subject through a hypodermic needle bya syringe in some embodiments of the invention. In various embodiments,cells are administered to the subject through a catheter. In a varietyof embodiments, cells are administered by surgical implantation. Furtherin this regard, in various embodiments, cells are administered to thesubject by implantation using an arthroscopic procedure. In someembodiments cells are administered to the subject in or on a solidsupport, such as a polymer or gel. In various embodiments, cells areadministered to the subject in an encapsulated form.

In additional embodiments of the invention, cells are suitablyformulated for oral, rectal, epicutaneous, ocular, nasal, and/orpulmonary delivery and are administered accordingly.

Compositions can be administered in dosages and by techniques well knownto those skilled in the medical and veterinary arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient, and the formulation that will be administered (e.g.,solid vs. liquid). Doses for humans or other mammals can be determinedwithout undue experimentation by the skilled artisan, from thisdisclosure, the documents cited herein, and the knowledge in the art.

Kits

In some embodiments, the disclosure provides kits for the production ofinduced pluripotent stem cells. In some embodiment, the kits include aDot1L inhibitor. In some embodiments, the kits include a Dot1L inhibitorand a reprogramming cocktail (e.g., Sox2 and/or Oct4). In someembodiments, the kits include instructions for the production of inducedpluripotent stem cells. In some embodiment, the kits include a YY1inhibitor. In some embodiment, the kits include an SUV39H1 inhibitor.

In some embodiments, the disclosure provides a kit for the production ofinduced pluripotent stem cells. In some embodiments, the kit includesone or more Dot1L inhibitors. In some embodiments, the Dot1L inhibitoris a compound of Formula I, II, III or IV (for example one or more ofthe embodiments described herein), or a pharmaceutically acceptable saltthereof. In some embodiments, the Dot1L inhibitor is an expressioninhibitor such as an RNAi inhibitor, for example one or more of theshRNA molecules provided herein.

In some embodiments, the kit includes one or more YY1 inhibitors and/orone or more SUV39H1 inhibitors. In some embodiments, the YY1 and/orSUV39H1 inhibitors are small molecule inhibitors or expressioninhibitors such as RNAi inhibitors, for example one or more of the shRNAmolecules provided herein.

In some embodiments, the kits include a reprogramming cocktail, or oneor more moieties that can be used to generate a reprogramming cocktail.In some embodiments, the kit includes nucleic acids for the introductionof one or more stem cell-associated genes into cells, including, forexample Oct3/4 (Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1,Klf2, Klf4, Klf5, c-Myc, 1-Myc, n-Myc and LIN28. In some embodiments,the kit includes nucleic acids for the introduction of Oct-4, Sox2,c-MYC, and Klf4 into a cell. In some embodiments, the kit includesnucleic acids for the introduction of Oct-4, and Sox2 into a cell. Insome embodiments, the nucleic acids are in delivery vehicle such as aviral vector, such as an adenoviral vector, a lentiviral vector or aretroviral vector.

In some embodiments, this kit includes one or more agents that enhancereprogramming efficiency including, for example, soluble Wnt, Wntconditioned media, BIX-01294 (a G9a histone methyltransferase),PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histonedeacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine,dexamethasone, suberoylanilide, hydroxamic acid (SAHA), trichostatin(TSA), and inhibitors of the TGF-β signaling pathway.

In some embodiments, this kit includes one or more agents that can bindmarkers to detect the presence of induced pluripotent stem cells. Suchmarkers include SSEA4, SSEA3, Tra-1-81, Oct4, Sox2 and Nanog. Agentsthat bind such markers include antibodies and compounds that selectivelybind the markers.

In some embodiments, the kit includes one or more elements useful inestablishing a reprogramming cocktail, including buffers, salts, sugars,and other components that may be useful to support the growth andreprogramming of the differentiated cells.

In some embodiments, the kit includes one or more components foradministering the induced pluripotent stem cells or differentiatedcells. These components include pharmaceutical carriers for systemicand/or local administration of the cells.

In some embodiments, the kit includes separate containers. Suchcontainers include small glass containers, plastic containers or stripsof plastic or paper. Such containers allow the efficient transfer ofreagents from one compartment to another compartment such that thesamples and reagents are not cross-contaminated and the agents orsolutions of each container can be added in a quantitative fashion fromone compartment to another.

Dot1L

In one aspect, the disclosure provides methods of promoting theproduction of induced pluripotent stem cells. In some embodiments, themethods of producing induced pluripotent stem cells include inhibitingDot1L in a differentiated cell. In some embodiments, the differentiatedcell is cultured under reprogramming conditions to produce inducedpluripotent stem cells. In some embodiments, the methods of producinginduced pluripotent stem comprise inhibiting the methyltransferaseactivity of Dot1L.

In some embodiments, the methods of producing induced pluripotent stemcomprise inhibiting Dot1L in a differentiated cell. Dot1L is a histonemethyl transferase (HMT) known to methylate lysine 79 of histone H3(“H3K79”) in viveo (Feng et al. (2002) Curr. Biol. 12: 1052-1058).Similar to other HMTs, Dot1L contains a 5-adenosylmethionine (SAM)binding site and uses SAM as a methyl donor. Dot1L nucleic acid andpolypeptides have previously been described (see, e.g., U.S. PatentApplication Publication No. 2005-0048634 A1 (incorporated by reference);Feng et al. (2002) Curr. Biol. 12: 1052-1058; and Okada et al. (2005)Cell 121: 167-78). The human Dot1L homolog has been cloned, isolated,and has been designated as hDot1L (human Dot1-like protein). Thesequences of the human nucleic acid and protein have been depositedunder GenBank Accession No. AF509504, while the mouse homolog is GenBankAccession No. XP125730). Additional hDot1L homologs are known as well(See e.g., WO2012075500). The 2.5 angstrom resolution structure of afragment of the hDot1L protein containing the catalytic domain (aminoacids 1-416) has been solved; and the atomic coordinates for amino acids1-416 of hDot1L have been determined and deposited in the RCSB databaseunder ID code 1NW3 and described in the scientific literature (see Min,et al. (2003) Cell 112:711-723).

In some embodiments, Dot1L is inhibited by contacting Dot1l, or a cellexpressing Dot1L with one or more of the compounds provided herein. Insome embodiments, Dot1l is inhibited by contacting a cell expressingDot1l with a nuclei acid that “knocks down” Dot1L, thereby inhibitingDot1L. It should be appreciated that inhibiting Dot1L activity as usedherein included both complete (i.e., about 100%) and partial inhibition.In some embodiments, partial inhibition results in 90% or less, 80% orless, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less,20% or less, 10% or less, 5% or less, 2% or less, or 1% or less up tocomplete inhibition of Dot1L. Dot1L inhibition in a cell can be achievedboth by suppressing one more biological functions of Dot1L (e.g., bycontacting the cell with a compound that binds the active site ofDot1L), or by “knocking down” Dot1L (e.g., by contacting the cell with anucleic acid that prevents production of Dot1L in the cell. In someembodiments, Dot1L is inhibited by inhibiting the catalytic function ofDot1L.

In one aspect, the disclosure provides methods of producing inducedpluripotent stem comprise inhibiting Dot1L in a differentiated cell. Insome embodiments inhibiting Dot1L comprises inhibiting themethyltransferase activity of Dot1L. In some embodiments, Dot1L isinhibited by contacting the cell with one or more small molecules thatinhibit Dot1L activity. Small molecules that inhibit Dot1L are describedfor instance in WO2012/075500, WO2012/082436, WO2012/075381, andWO2012/075492.

In some embodiments, Dot1L is inhibited by contacting the differentiatedcell with a composition comprising a compound of formula I:

or a pharmaceutically acceptable salt, hydrate, enantiomer orstereoisomer thereof, wherein independently for each occurrence,

X is

R¹ is hydrogen, alkyl, cycloalkyl, alkylcycloalkyl, alkylaryl,haloalkyl, formyl, heterocyclyl, heterocyclylalkyl,

or (C₂-C₄) alkyl substituted with

except that when X is

R¹ is not

R¹⁰ is hydrogen or alkyl;

R^(11a) is hydrogen, alkyl, or alkyl-cycloalkyl;

R^(11b) is hydrogen or alkyl; or taken together with R^(11a) and thenitrogen to which it is attached forms a 4- to 8-membered heterocyclylcomprising 0 or 1 additional heteroatoms;

R¹³ is hydrogen, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, orsilyl;

R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R¹⁵ is alkyl, cycloalkyl, or cycloalkylalkyl;

R²⁰ is hydrogen, alkyl, cycloalkyl, or cycloalkylalkyl;

R² is

Y is —NH—, —N(alkyl)-, —O—, or —CR⁶ ₂—;

R^(22a) is aryl, heteroaryl, aralkyl, heteroaralkyl, fused bicyclyl,biaryl, aryloxyaryl, heteroaryloxyaryl, aryloxyheteroaryl, orheteroaryloxyheteroaryl;

R^(22b) is hydrogen or alkyl;

R²⁴ is hydrogen or alkyl;

R^(25a), R^(25b), R^(25c), and R^(25d) independently are -M₂-T₂, inwhich M₂ is a bond, SO₂, SO, S, CO, CO₂, O, O—C₁-C₄ alkyl linker, C₁-C₄alkyl linker, NH, or N(R_(t)), R_(t) being C₁-C₆ alkyl, and T₂ is H,halo, or R_(S4), R_(S4) being C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 8-membered heterocycloalkyl, or 5 to10-membered heteroaryl, and each of O—C₁-C₄ alkyl linker, C₁-C₄ alkyllinker, R_(t), and Rs₄ being optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,carboxyl, cyano, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, and 5 to6-membered heteroaryl;

R³ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy,alkylcarbonyloxy, or silyloxy;

R⁴ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy,alkylcarbonyloxy, or silyloxy;

R⁴¹ is hydrogen, alkyl, or alkynyl;

Z is hydrogen or

R^(5a) is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,aralkylthioalkyl, or heteroaralkylthioalkyl; or alkyl substituted with1, 2 or 3 substituents independently selected from the group consistingof hydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino,amido, aryl, and heteroaryl;

R^(5b) is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,aralkylthioalkyl, or heteroaralkylthioalkyl; or alkyl substituted with1, 2 or 3 substituents independently selected from the group consistingof hydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino,amido, aryl, and heteroaryl; or taken together with R^(5a) and thenitrogen to which it is attached forms a 4- to 8-membered heterocyclylcomprising 0 or 1 additional heteroatoms;

R⁶ is hydrogen, alkyl, or halo; or two geminal R⁶ taken together areethylene, propylene, or butylene;

R^(7a) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2, or 3substituents independently selected from the group consisting of cyano,lower alkoxy, and halo;

R^(7b) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2, or 3substituents independently selected from the group consisting of cyano,lower alkoxy, and halo; and

R^(7c) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2, or 3substituents independently selected from the group consisting of cyano,lower alkoxy, and halo.

Compounds of formula I, methods of making thereof, and methods of usethereof can be found in WO 2012/075500, which is incorporated herein byreference.

In certain embodiments, X is

In certain embodiments, X is

In certain embodiments,

In certain embodiments, R² is

In certain embodiments, R²⁴ is hydrogen or alkyl. In certainembodiments, R²⁴ is hydrogen.

In certain embodiments, R^(25a) is hydrogen, alkyl, —O-alkyl, halogen,trifluoroalkyl, —O— trifluoromethyl, or —SO₂-trifluoromethyl. In certainembodiments, R^(25b) is hydrogen, alkyl, halogen, or trifluoroalkyl. Incertain embodiments, R^(25c) is hydrogen, alkyl, or halogen. In certainembodiments, R^(25c) is hydrogen or halogen.

In certain embodiments, R² is

In certain embodiments, Y is —NH— or —N(alkyl)-. In certain embodiments,Y is —NH—.

In certain embodiments, Y is —N(CH₃)—. In certain embodiments Y is —O—.In certain embodiments, Y is —CH₂—.

In certain embodiments, R^(22a) is aryl or aralkyl. In certainembodiments, R^(22a) is substituted phenyl or substituted benzyl. Incertain embodiments, R^(22a) is one of the following:

In certain embodiments, R^(22a) is one of the following:

In certain embodiments, R^(22b) is hydrogen. In certain embodiments,R^(22b) is methyl.

In certain embodiments, R¹ is hydrogen. In certain embodiments, R¹ isalkyl. In certain embodiments, R¹ is —CH₃, —CH₂CH₃, —CH₂CH(CH₃)₂ or—CH₂CH₂CH(CH₃)₂. In certain embodiments, R¹ is C₃-C₇ cycloalkyl. Incertain embodiments, R¹ is cyclopropyl, cyclopropylmethyl,2-cyclopropylethyl, cyclobutyl, cyclobutylmethyl, 2-cyclobutylethyl,cyclopentyl, cyclopentylmethyl, or 2-cyclopentylethyl. In certainembodiments, R¹ is

In certain embodiments, R¹ is —CH₂CF₃. In certain embodiments, R¹ is—CH₂Ph. In certain embodiments, R¹ is —C(═O)H. In certain embodiments,R¹ is —C(═O)CH₃. In certain embodiments, R¹ is heterocyclyl orheterocyclylalkyl. In certain embodiments, R¹ is

In certain embodiments, R¹ is

In certain embodiments, R¹⁵ is alkyl. In certain embodiments, R¹⁵ iscycloalkyl. In certain embodiments, R¹⁵ is cycloalkylalkyl.

In certain embodiments, R¹ is (C₂-C₄) alkyl substituted with

In certain embodiments, R¹ is

In certain embodiments, R^(11a) is hydrogen, alkyl, or alkyl-cycloalkyl.In certain embodiments, R^(11a) is hydrogen, methyl, or i-propyl. Incertain embodiments, R¹ is

In certain embodiments, R¹³ is hydrogen.

In certain embodiments, R¹ is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

and R⁶ is alkyl. In certain embodiments, A is

and R⁶ is methyl, ethyl, or isopropyl.

In certain embodiments, R³ is hydroxyl. In certain embodiments, R³ ishydrogen. In certain embodiments, R⁴ is hydroxyl. In certainembodiments, R is hydrogen. In certain embodiments, R⁴¹ is hydrogen. Incertain embodiments, R⁴¹ is methyl. In certain embodiments, R³ ishydroxyl; and R is hydroxyl. In certain embodiments, R³ is hydroxyl; R⁴is hydroxyl; and R⁴¹ is hydrogen. In certain embodiments, R³ ishydroxyl; R⁴ is hydroxyl; and R⁴¹ is methyl. In certain embodiments, R³is hydrogen; and R⁴ is hydroxyl. In certain embodiments, R³ is hydrogen;R⁴ is hydroxyl; and R⁴¹ is hydrogen. In certain embodiments, R³ ishydrogen; R⁴ is hydroxyl; and R⁴¹ is methyl. In certain embodiments, R³is hydroxyl; and R⁴ is hydrogen. In certain embodiments, R³ is hydroxyl;R⁴ is hydrogen; and R⁴ is hydrogen. In certain embodiments R³ ishydroxyl; R⁴ is hydrogen; and R⁴¹ is methyl.

In certain embodiments, Z is hydrogen or

In certain embodiments, Z is hydrogen. In certain embodiments, Z is

In certain embodiments, R^(5a) is hydrogen, alkyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, carbocyclylalkyl, heterocyclylalkyl,aralkyl, or heteroaralkyl. In certain embodiments, R^(5a) is hydrogen,aralkyloxyalkyl, alkyl, aryl, aralkyl, aminoalkyl or hydroalkyl. Incertain embodiments, R^(5a) is —H, —CH₂CH₂OCH₂Ph, —CH₂CH₃, —CH(CH₃)₂,-Ph, —CH₂CH(CH₃), —CH₃, —CH₂Ph, —CH₂CH₂NH₂, —CH₂(cyclohexyl) or—CH₂CH₂OH. In certain embodiments, R^(5b) is hydrogen, alkyl,carbocyclyl, heterocyclyl, aryl, heteroaryl, carbocyclylalkyl,heterocyclylalkyl, aralkyl, or heteroaralkyl. In certain embodiments,R^(5b) is hydrogen, aralkyloxyalkyl, alkyl, aryl, aralkyl, aminoalkyl orhydroalkyl. In certain embodiments, R^(5b) is hydrogen. In certainembodiments, R^(5a) is —H, —CH₂CH₂OCH₂Ph, —CH₂CH₃, —CH(CH₃)₂, -Ph,—CH₂CH(CH₃), —CH₃, —CH₂Ph, —CH₂CH₂NH₂, —CH₂(cyclohexyl) or —CH₂CH₂OH;and R^(5b) is —H.

In certain embodiments, R^(7a) is hydrogen or lower alkyl. In certainembodiments, R^(7a) is hydrogen. In certain embodiments, R^(7b) ishydrogen or lower alkyl. In certain embodiments, R^(7b) is hydrogen. Incertain embodiments, R^(7c) is hydrogen or lower alkyl. In certainembodiments, R^(7c) is hydrogen.

In some embodiments, Dot1L is inhibited by contacting the differentiatedcell with a composition comprising a compound of formula II:

or a pharmaceutically acceptable salt, hydrate, enantiomer orstereoisomer thereof, wherein independently for each occurrence,

X is

R¹ is hydrogen, alkyl, cycloalkyl, alkylcycloalkyl, alkylaryl,haloalkyl, formyl, heterocyclyl, heterocyclylalkyl,

or (C₂-C₄)alkyl substituted with

except that when X is

R¹ is not

R¹⁰ is hydrogen or alkyl;

R^(11a) is hydrogen, alkyl, or alkyl-cycloalkyl;

R^(11b) is hydrogen or alkyl; or taken together with R and the nitrogento which it is attached forms a 4- to 8-membered heterocyclyl comprising0 or 1 additional heteroatoms;

R¹³ is hydrogen, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl orsilyl;

R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R¹⁵ is alkyl, cycloalkyl or cycloalkylalkyl;

R²⁰ is hydrogen, alkyl, cycloalkyl or cycloalkylalkyl;

A is

R² is

Y is —NH—, —N(alkyl)-, —O—, or —CR⁶ ₂;

R^(22a) is aryl, heteroaryl, aralkyl, heteroaralkyl, fused bicyclyl,biaryl, aryloxyaryl, heteroaryloxyaryl, aryloxyheteroaryl orheteroaryloxyheteroaryl;

R^(22b) is hydrogen or alkyl;

R²⁴ is hydrogen or alkyl;

R^(25a), R^(25b), R^(25c), R^(25d) are independently -M₂-T₂, in which M₂is a bond, SO₂, SO, S, CO, CO₂, O, O—C₁-C₄ alkyl linker, C₁-C₄ alkyllinker, NH, or N(R_(t)), R_(t) being C₁-C₆ alkyl, and T₂ is H, halo, orRs₄, Rs₄ being C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, biaryl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 8-membered heterocycloalkyl, or 5 to10-membered heteroaryl, and each of O—C₁-C₄ alkyl linker, C₁-C₄ alkyllinker, R_(t), and Rs₄ being optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,carboxyl, cyano, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁—C alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, and 5 to6-membered heteroaryl;

R³ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxyor silyloxy;

R⁴ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxyor silyloxy;

R⁴¹ is hydrogen, alkyl or alkynyl;

Z is hydrogen or

R^(5a) is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl, carbocyclylalkyl,heterocyclylalkyl, aralkyl, heteroaralkyl, alkylcarbonylarhinoalkyl,arylcarbonylaminoalkyl, aralkylcarbonylaminoalkyl,arylsulfonylaminoalkyl, alkylthioalkyl, aralkylthioalkyl orheteroaralkylthioalkyl; or alkyl substituted with 1, 2 or 3 substituentsindependently selected from the group consisting of hydroxy, halo,carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino, amido, aryl andheteroaryl;

R^(5b) is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl, carbocyclylalkyl,heterocyclylalkyl, aralkyl, heteroaralkyl, alkylcarbonylaminoalkyl,arylcarbonylaminoalkyl, aralkylcarbonylaminoalkyl,arylsulfonylaminoalkyl, alkylthioalkyl, aralkylthioalkyl orheteroaralkylthioalkyl; or alkyl substituted with 1, 2 or 3 substituentsindependently selected from the group consisting of hydroxy, halo,carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino, amido, aryl andheteroaryl; or taken together with R^(5a) and the nitrogen to which itis attached forms a 4- to 8-membered heterocyclyl comprising 0 or 1additional heteroatoms;

R⁶ is hydrogen, alkyl or halo; or two geminal R⁶ taken together areethylene, propylene or butylene; and

R^(7a) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2, or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo; and

R^(7b) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2, or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo.

Compounds of formula II, methods of making thereof, and methods of usethereof can be found in WO 2012/082436, which is incorporated herein byreference.

In certain embodiments, X is

In certain embodiments, X is

In certain embodiments, R² is

In certain embodiments, R² is

In certain embodiments, R²⁴ is hydrogen or alkyl. In certainembodiments, R²⁴ is hydrogen. In certain embodiments, R^(25a) ishydrogen, alkyl, —O-alkyl, halogen, trifluoroalkyl, —O-trifluoromethyl,or —SO₂-trifluoromethyl. In certain embodiments, R^(25b) is hydrogen,alkyl, halogen, or trifluoroalkyl. In certain embodiments, R^(25c) ishydrogen, alkyl, or halogen. In certain embodiments, R^(25c) is hydrogenor halogen.

In certain embodiments, R² is

In certain embodiments, Y is —NH— or —N(alkyl)-. In certain embodiments,Y is —NH—. In certain embodiments, Y is —N(CH₃)—. In certainembodiments, Y is —O—. In certain embodiments, Y is —CH₂—. In certainembodiments, R^(22a) is aryl or aralkyl. In certain embodiments, R^(22a)is substituted phenyl or substituted benzyl. In certain embodiments,R^(22a) is one of the following:

In certain embodiments, R^(22a) is one of the following:

In certain embodiments, R^(22b) is hydrogen. In certain embodiments,R^(22b) is methyl.

In certain embodiments, R¹ is hydrogen. In certain embodiments, R¹ isalkyl. In certain embodiments, R¹ is —CH₃, —CH₂CH₃, —CH₂CH(CH₃)₂ or—CH₂CH₂CH(CH₃)₂. In certain embodiments, R¹ is

In certain embodiments, R is —CH₂CF₃. In certain embodiments, R¹ is—CH₂Ph. In certain embodiments, R¹ is —C(═O)H. In certain embodiments,R¹ is —C(═O)CH₃. In certain embodiments, R¹ is heterocyclyl orheterocyclylalkyl. In certain embodiments, R¹ is

In certain embodiments, R¹ is

In certain embodiments, R¹ is

In certain embodiments, R¹ is

In certain embodiments, R¹⁵ is alkyl. In certain embodiments, R¹⁵ ismethyl. In certain embodiments, R¹⁵ is cycloalkyl. In certainembodiments, R¹⁵ is cycloalkylalkyl.

In certain embodiments, R¹ is (C₂-C₄)alkyl substituted with

In certain embodiments, R¹ is

In certain embodiments, R^(11a) is hydrogen, alkyl, or alkyl-cycloalkyl.In certain embodiments, R^(11a) is hydrogen, methyl, or i-propyl. Incertain embodiments, R^(22a) is heteroaryl. In certain embodiments,R^(22a) is substituted phenyloxyphenyl, substituted 4-(phenyl)phenyl oroptionally substituted 4-(heteroaryl)phenyl.

In certain embodiments, R^(22a) is one of the following:

In certain embodiments, R^(22a) is one of the following:

In certain embodiments, R^(11a) is hydrogen. In certain embodiments,R^(11b) is hydrogen. In certain embodiments, R^(11b) is methyl.

In certain embodiments, R¹ is

In certain embodiments, R¹ is

In certain embodiments, R¹³ is hydrogen.

In certain embodiments, R¹ is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

In certain embodiments, A is

and R⁶ is alkyl. In certain embodiments, A is

and R⁶ is methyl, ethyl, or isopropyl.

In certain embodiments, R³ is hydroxyl. In certain embodiments, R³ ishydrogen. In certain embodiments, R⁴ is hydroxyl. In certainembodiments, R⁴ is hydrogen. In certain embodiments, R⁴¹ is hydrogen. Incertain embodiments, R⁴¹ is methyl. In certain embodiments, R³ ishydroxyl; and R⁴ is hydroxyl. In certain embodiments, R³ is hydroxyl; R⁴is hydroxyl; and R⁴¹ is hydrogen. In certain embodiments, R³ ishydroxyl; R⁴ is hydroxyl; and R⁴¹ is methyl. In certain embodiments, R³is hydrogen; and R⁴ is hydroxyl. In certain embodiments, R³ is hydrogen;R⁴ is hydroxyl; and R⁴¹ is hydrogen. In certain embodiments R³ ishydrogen; R⁴ is hydroxyl; and R⁴¹ is methyl. In certain embodiments, R³is hydroxyl; and R⁴ is hydrogen. In certain embodiments, R³ is hydroxyl;R⁴ is hydrogen; and R⁴¹ is hydrogen. In certain embodiments, R³ ishydroxyl; R⁴ is hydrogen; and R⁴¹ is methyl.

In certain embodiments, Z is hydrogen or

In certain embodiments, Z is hydrogen. In certain embodiments, Z is

In certain embodiments, R^(5a) is hydrogen, alkyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, carbocyclylalkyl, heterocyclylalkyl,aralkyl, or heteroaralkyl. In certain embodiments, R^(5a) is hydrogen,aralkyloxyalkyl, alkyl, aryl, aralkyl, aminoalkyl or hydroxyalkyl. Incertain embodiments, R^(5a) is —H, —CH₂CH₂OCH₂Ph, —CH₂CH₃, —CH(CH₃)₂,-Ph, —CH₂CH(CH₃), —CH₃, —CH₂Ph, —CH₂CH₂NH₂, —CH₂(cyclohexyl) or—CH₂CH₂OH. In certain embodiments, R^(5b) is hydrogen, alkyl,carbocyclyl, heterocyclyl, aryl, heteroaryl, carbocyclylalkyl,heterocyclylalkyl, aralkyl, or heteroaralkyl. In certain embodiments,R^(5b) is hydrogen, aralkyloxyalkyl, alkyl, aryl, aralkyl, aminoalkyl orhydroalkyl. In certain embodiments, R^(5b) is hydrogen. In certainembodiments, R^(5a) is —H, —CH₂CH₂OCH₂Ph, —CH₂CH₃, —CH(CH₃)₂, -Ph,—CH₂CH(CH₃), —CH₃, —CH₂Ph, —CH₂CH₂NH₂, —CH₂(cyclohexyl) or —CH₂CH₂OH;and R^(5b) is —H.

In certain embodiments, R^(7a) is hydrogen or lower alkyl. In certainembodiments, R^(7a) is hydrogen. In certain embodiments, R^(7b) ishydrogen or lower alkyl. In certain embodiments, R^(7b) is hydrogen.

In some embodiments, Dot1L is inhibited by contacting the differentiatedcell with a composition comprising a compound of formula III:

or pharmaceutically acceptable salt or ester thereof, wherein:

A is O or CH₂;

each of G and J, independently, is H, halo, C(O)OH, C(O)O—C₁-C₆ alkyl orOR_(a), R_(a) being H, C₁-C₆ alkyl or C(O)—C₁-C₆ alkyl, whereinC(O)O—C₁-C₆ alkyl, C₁-C₆ alkyl or C(O)—C₁-C₆ alkyl is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, cyano, hydroxyl, carboxyl, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, and C₃-C₈ cycloalkyl;

Q is H, NH₂, NHR_(b), NR_(b)R_(c), R_(b), or OR_(b), in which each ofR_(b) and R_(c) independently is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 7-memberedheterocycloalkyl, 5 to 10-membered heteroaryl, or -M₁-T₁ in which M₁ isa bond or C₁-C₆ alkyl linker optionally substituted with halo, cyano,hydroxyl or C₁-C₆ alkoxyl and T₁ is C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to6-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, or R_(b)and R_(c), together with the N atom to which they attach, form 4 to7-membered heterocycloalkyl having 0 or 1 additional heteroatoms to theN atom optionally substituted with C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, halo, hydroxyl, carboxyl, C(O)OH, C(O)O—C₁-C₆ alkyl,OC(O)—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, or 5 to 6-membered heteroaryl, and each of R_(b),R_(c), and T₁ is optionally substituted with one or more substituentsselected from the group consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, halo, hydroxyl, carboxyl, cyano, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;

X is N or CR_(X), in which R_(x) is H, halo, hydroxyl, carboxyl, cyano,or Rs₁, Rs₁ being amino, C₁-C₆ alkoxyl, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, or 5 to 6-membered heteroaryl, and R_(S1) beingoptionally substituted with one or more substituents selected from thegroup consisting of halo, hydroxyl, carboxyl, cyano, C₁-C₆ alkoxyl,amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-memberedheteroaryl;

L₁ is N(Y), S, SO, or SO₂;

L₂ is CO or absent when L₁ is N(Y) or is absent when L₁ is S, SO, orSO₂, in which Y is H, R_(d), SO₂R_(d), or COR_(d) when L₂ is absent, orY is H or R_(d) when L₂ is CO, R_(d) being C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, or 5 to 6-membered heteroaryl, and R_(d) beingoptionally substituted with one or more substituents selected from thegroup consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, halo,hydroxyl, carboxyl, cyano, C₁-C₆ alkoxyl, C₁-C₆ alkylsulfonyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryland with C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, or 5 to 6-membered heteroaryl further optionallysubstituted with C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, halo,hydroxyl, carboxyl, C(O)OH, C(O)O—C₁-C₆ alkyl, OC(O)—C₁-C₆ alkyl, cyano,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, or 5 to6-membered heteroaryl;

each of R₁, R₂, R₃, R⁴, R₅, R₆, and R₇, independently, is H, halo,hydroxyl, carboxyl, cyano, Rs₂, Rs₂ being amino, C₁-C₆ alkoxyl, C₁-C₆alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, and each Rs₂ being optionallysubstituted with one or more substituents selected from the groupconsisting of halo, hydroxyl, carboxyl, cyano, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;

R₈ is H, halo or Rs₃, Rs₃ being C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl, and Rs₃ being optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,carboxyl, cyano amino, C₁-C₆ alkoxyl, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, and C₃-C₈ cycloalkyl;

R⁹ is

in which each of R_(c), R_(f), R_(g), and R_(h), independently is-M₂-T₂, in which M₂ is a bond, SO₂, SO, S, CO CO₂, O, O—C₁-C₄ alkyllinker, C₁-C₄ alkyl linker, NH, or N(R_(t)), R_(t) being C₁-C₆ alkyl,and T₂ is H, halo, or R_(S4), Rs₄ being C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 8-memberedheterocycloalkyl, or 5 to 10-membered heteroaryl, and each of O—C₁-C₄alkyl linker, C₁-C₄ alkyl linker, R_(t), and R_(S4) being optionallysubstituted with one or more substituents selected from the groupconsisting of halo, hydroxyl, carboxyl, cyano, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, and 5 to 6-membered heteroaryl, R_(i) is H or C₁-C₆alkyl optionally substituted with one or more substituents selected fromthe group consisting of halo, hydroxyl, carboxyl, cyano, C₁-C₆ alkoxyl,amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-memberedheteroaryl, D is O, NR_(j), or CR_(j)R_(k), each of R_(j) and R_(k)independently being H or C₁-C₆ alkyl, or R_(j) and R_(k) taken together,with the carbon atom to which they are attached, form a C₃-C₁₀cycloalkyl ring, and E is -M₃-T₃, M₃ being a bond or C₁-C₆ alkyl linkeroptionally substituted with halo or cyano, T₃ being C₃-C₁₀ cycloalkyl,C₆-C₁₀ aryl, 5 to 10-membered heteroaryl, or 4 to 10-memberedheterocycloalkyl, and T₃ being optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,thiol, carboxyl, cyano, nitro, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ alkoxyl, C₁-C₆ haloalkyl, C₁-C₆ haloalkoxyl, C₁-C₆alkylthio, C₁-C₆ alkylsulfonyl, C₁-C₆ haloalkylsulfonyl, C₁-C₆alkylcarbonyl, C₁-C₆ alkoxycarbonyl, oxo, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₄-C₁₂ alkylcycloalkyl, C₆-C₁₀aryl, C₆-C₁₀ aryloxyl, C₇-C₁₄ alkylaryl, C₆-C₁₀ aminoaryloxyl, C₆-C₁₀arylthio, 4 to 6-membered heterocycloalkyl optionally substituted withhalo, C₁-C₄ alkyl, C₁-C₄ haloalkyl, 5 to 6-membered heteroaryloptionally substituted with halo, C₁-C₄ alkyl, and C₁-C₆ alkyl that issubstituted with hydroxy, halo, C₁-C₆ alkoxycarbonyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-memberedheteroaryl optionally further substituted with halo, hydroxyl, or C₁-C₆alkoxyl;

q is 0, 1, 2, 3, or 4;

m is 0, 1, or 2; and

n is 0, 1, or 2.

Compounds of formula III, methods of making thereof, and methods of usethereof can be found in WO 2012/075500, which is incorporated herein byreference.

In certain embodiments, the sum of m and n is at least 1. In certainembodiments, m is 1 or 2 and n is 0. In certain embodiments, m is 2 andn is 0.

In certain embodiments, A is CH₂. In certain embodiments, A is O.

In certain embodiments, L₁ is N(Y). In certain embodiments, L₁ is SO orSO₂.

In certain embodiments, Y is Rj.

In certain embodiments, R_(d) is C₁-C₆ alkyl.

In certain embodiments, L₂ is absent.

In certain embodiments, each of G and J independently is OR. In certainembodiments, R_(a) is hydrogen.

In certain embodiments, R₉ is

In certain embodiments, R₉ is

In certain embodiments, at least one of R_(e), R_(f), R_(g), and R_(h)is halo (such as F, CI, and Br), C₁-C₆ alkoxyl optionally substitutedwith one or more halo (such as OCH₃, OCH₂CH₃, O-iPr, and OCF₃), C₁-C₆alkylsulfonyl optionally substituted with one or more halo (such asSO₂CF₃), or C₁-C₆ alkyl optionally substituted with one or more halo(such as CH₃, i-propyl, n-butyl, and CF₃). In certain embodiments, Rj isH or C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl).

In certain embodiments,

is unsubstituted benzimidazolyl or one of the following groups:

In certain embodiments, R⁹ is

In certain embodiments, D is O. In certain embodiments, D is NR_(j). Incertain embodiments, R_(j) is H. In certain embodiments, D is CRjR_(k).In certain embodiments, each of R_(j) and R_(k) is hydrogen. In certainembodiments, E is -M₃-T₃, in which M₃ is a bond or C₁-C₃ alkyl linker,T₃ is phenyl, naphthyl, thienyl, cyclopropyl, or cyclohexyl, and T₃ isoptionally substituted with one or more substituents selected from thegroup consisting of halo, hydroxyl, thiol, carboxyl, cyano, nitro, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxyl, C₁-C₆ haloalkyl,C₁-C₆ haloalkoxyl, C₁-C₆ alkylthio, C₁-C₆ alkylsulfonyl, C₁-C₆alkylcarbonyl, C₁-C₆ alkoxycarbonyl, oxo, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₄-C₁₂ alkylcycloalkyl, C₆-C₁₀aryl, C₆-C₁₀ aryloxyl, C₇-C₁₄ alkylaryl, C₆-C₁₀ aminoaryloxyl, C₆-C₁₀arylthio, 4 to 6-membered heterocycloalkyl optionally substituted withC₁-C₄ alkyl, 5 to 6-membered heteroaryl optionally substituted withC₁-C₄ alkyl, and C₁-C₆ alkyl that is substituted with hydroxy, C₁-C₆alkoxycarbonyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, or 5 to 6-membered heteroaryl. In certain embodiments,T₃ is phenyl optionally substituted with one or more substituentsselected from the group consisting of halo, hydroxyl, carboxyl, cyano,nitro, C₁-C₆ alkyl (e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl), C₁-C₆ alkoxyl, C₁-C₆haloalkyl, C₁-C₆ haloalkoxyl, C₁-C₆ alkylsulfonyl, C₆-C₁₀ aryl (e.g.,phenyl or naphthyl), and C₆-C₁₀ aryloxyl, and C₇-C₁₄ alkylaryl.

In certain embodiments, E is

In certain embodiments, X is N. In certain embodiments, X is CR_(X). Incertain embodiments, X is CH.

In certain embodiments, Q is NH₂ or NHR_(b), in which R_(b) is -M₁-T₁,M₁ being a bond or C₁-C₆ alkyl linker and T₁ being C₃-C₈ cycloalkyl. Incertain embodiments, Q is H.

In certain embodiments, R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are each H.

In certain embodiments, when R₈ is halo and is attached to the samecarbon atom as J, then J is not hydroxyl. In certain embodiments, whenR₈ is halo and is attached to the same carbon atom as G, then G is nothydroxyl. In certain embodiments, T₂ is not halo when M₂ is SO₂, SO, S,CO or O.

In certain embodiments, T₂ is a 4-8 membered heterocycloalkyl which isbound to M₂ via a heteroatom. In certain embodiments, T₂ is a 4-8membered heterocycloalkyl which is bound to M₂ via a N atom. In certainembodiments, T₂ is a 4-8 membered heterocycloalkyl which is bound to M₂via a C atom.

In certain embodiments, the compound is of formula III-a:

or pharmaceutically acceptable salt or ester thereof, wherein A, Q, X,L_(I), L₂, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, q, m, and n are asdescribed herein for formula III.

In certain embodiments, the compound is of formula III-b or III-c:

or pharmaceutically acceptable salt or ester thereof, wherein A, Q, X,L₁, L₂, R₁, R₂, R₃, R₄, R₅, R⁶, R₇, R₈, R_(e), R_(f), R_(g), R_(h),R_(i), q, m, and n are as described herein for formula III.

In certain embodiments, the compound is of formula III-d:

or pharmaceutically acceptable salt or ester thereof, wherein A, Q, X,G, J, L₁, L₂, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, D, E, q, m, and n are asdescribed herein for formula III.

In certain embodiments, the compound is of formula III-e:

or its N-oxide, or a pharmaceutically acceptable salt or ester thereof,wherein A, Q, X, Y, R₁, R₂, R_(e), R_(f), R_(g), R_(h), and m are asdescribed herein for formula III.

In certain embodiments, A is O. In certain embodiments, A is O and m is2. In certain embodiments, X is N. In certain embodiments, Q is NH₂ orNHR_(b), in which R_(b) is -M₁-T, M₁ being a bond or C₁-C₆ alkyl linkerand T₁ being C₃-C₈ cycloalkyl. In certain embodiments, R₁ and R₂ areeach H. In certain embodiments, Y is R_(d). In certain embodiments,R_(d) is C₁-C₆ alkyl optionally substituted with C₃-C₈ cycloalkyl orhalo. In certain embodiments, R_(d) is C₃-C₈ cycloalkyl optionallysubstituted with C₁-C₆ alkyl or halo. In certain embodiments, at leastone of R_(e), R_(f), R_(g), and R is halo, C₁-C₆ alkoxyl optionallysubstituted with one or more halo; C₁-C₆ alkylsulfonyl optionallysubstituted with one or more halo; C₁-C₆ alkyl optionally substitutedwith one or more substituents selected from CN, halo, C₃-C₈ cycloalkyl,hydroxy, and C₁-C₆ alkoxyl; C₃-C₈ cycloalkyl optionally substituted withone or more C₁-C₆ alkyl or CN; or 4 to 8-membered heterocycloalkyloptionally substituted with one or more substituents selected from CN,halo, hydroxy, C₁-C₆ alkyl and C₁-C₆ alkoxyl. In certain embodiments, atleast one of R_(e), R_(f), R_(g), and R_(h) is selected from F; Cl; Br;CF₃; OCF₃; SO₂CF₃; oxetanyl optionally substituted with one or moresubstituents selected from CN, halo, hydroxy, C₁-C₆ alkyl and C₁-C₆alkoxyl; C₃-C₈ cycloalkyl optionally substituted with one or moresubstituents selected from C₁-C₄ alkyl; and C₁-C₄ alkyl optionallysubstituted with one or more substituents selected from halo, C₃-C₈cycloalkyl, hydroxy and C₁-C₆ alkoxyl. In certain embodiments, at leastone of R_(f) and R_(g) is alkyl, optionally substituted with hydroxyl.In certain embodiments, at least one of R_(f) and R_(g) is i-butylsubstituted with hydroxyl.

In some embodiments, Dot1L is inhibited by contacting the differentiatedcell with a composition comprising a compound of formula IV:

or a pharmaceutically acceptable salt or ester thereof, wherein:

each of G and J, independently, is H, halo, C(O)OH, C(O)O—C₁-C₆ alkyl orOR_(a), R_(a), being H, C₁-C₆ alkyl or C(O)—C₁-C₆ alkyl, whereinC(O)O—C₁-C₆ alkyl, C₁-C₆ alkyl or C(O)—C₁-C₆ alkyl is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, cyano hydroxyl, carboxyl, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, and C₃-C₈ cycloalkyl;

Q is H, NH₂, NHR_(b), NR_(b)R_(c), R_(b), or OR_(b), in which each ofR_(b) and R_(c) independently is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 7-memberedheterocycloalkyl, 5 to 10-membered heteroaryl, or -M₁-T₁ in which M₁ isa bond or C₁-C₆ alkyl linker optionally substituted with halo, cyano,hydroxyl or C₁-C₆ alkoxyl and T_(t) is C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4to 6-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, or R_(b)and R_(c), together with the N atom to which they attach, form 4 to7-membered heterocycloalkyl having 0 or 1 additional heteroatoms to theN atom optionally substituted with C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, halo, hydroxyl, carboxyl, C(O)OH, C(O)O—C₁-C₆ alkyl,OC(O)—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, or 5 to 6-membered heteroaryl, and each of R_(b),R_(c), and T₁ is optionally substituted with one or more substituentsselected from the group consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, halo, hydroxyl, carboxyl, cyano, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;

X is N or CR_(X), in which R_(x) is H, halo, hydroxyl, carboxyl, cyano,or R_(S1), Rs₁ being amino, C₁-C₆ alkoxyl, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, or 5 to 6-membered heteroaryl, and Rs₁ beingoptionally substituted with one or more substituents selected from thegroup consisting of halo, hydroxyl, carboxyl, cyano, C₁-C₆ alkoxyl,amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-memberedheteroaryl;

L₁ is N(Y), S, SO, or SO₂;

L₂ is CO or absent when L₁ is N(Y) or is absent when L₁ is S, SO, orSO₂, in which Y is H, R_(d), SO₂R_(d), or COR_(d) when L₂ is absent, orY is H or R_(d) when L₂ is CO, R_(d) being C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, or 5 to 6-membered heteroaryl, and R_(d) beingoptionally substituted with one or more substituents selected from thegroup consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, halo,hydroxyl, carboxyl, cyano, C₁-C₆ alkoxyl, C₁-C₆ alkylsulfonyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryland with C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, or 5 to 6-membered heteroaryl further optionallysubstituted with C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, halo,hydroxyl, carboxyl, C(O)OH, C(O)O—C₁-C₆ alkyl, OC(O)—C₁-C₆ alkyl, cyano,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, or 5 to6-membered heteroaryl;

each of R₁, R₂, R₄, R₅, R⁶, and R₇, independently, is H, halo, hydroxyl,carboxyl, cyano, R_(S2), Rs₂ being amino, C₁-C₆ alkoxyl, C₁-C₆ alkyl,C₂-C₆ alkenyl, or C₂-C₆ alkynyl, and each Rs₂ being optionallysubstituted with one or more substituents selected from the groupconsisting of halo, hydroxyl, carboxyl, cyano, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;

R₈ is H, halo or Rs₃, Rs₃ being C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl, and Rs₃ being optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,carboxyl, cyano amino, C₁-C₆ alkoxyl, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, and C₃-C₈ cycloalkyl;

R₉ is

in which each of R_(e), R_(f), R_(g), and R_(h), independently is-M₂-T₂, in which M₂ is a bond, SO₂, SO, S, CO, CO₂, O, O—C₁-C₄ alkyllinker, C₁-C₄ alkyl linker, NH, or N(R_(t)), R being C₁-C₆ alkyl, and T₂is H, halo, or R_(S4), Rs₄ being C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 8-memberedheterocycloalkyl, or 5 to 10-membered heteroaryl, and each of O—C₁-C₄alkyl linker, C₁-C₄ alkyl linker, R_(t), and R_(S4) being optionallysubstituted with one or more substituents selected from the groupconsisting of halo, hydroxyl, carboxyl, cyano, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 6-memberedheterocycloalkyl, and 5 to 6-membered heteroaryl, R₁ is H or C₁-C₆ alkyloptionally substituted with one or more substituents selected from thegroup consisting of halo, hydroxyl, carboxyl, cyano, C₁-C₆ alkoxyl,amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-memberedheteroaryl; D is O, NR_(j), or CR_(j)R_(k), each of R_(j) and R_(k)independently being H or C₁-C₆ alkyl, or R_(j) and R_(k) taken together,with the carbon atom to which they are attached, form a C₃-C₁₀cycloalkyl ring, and E is-M₃-T₃, M₃ being a bond or C₁-C₆ alkyl linkeroptionally substituted with halo or cyano, T₃ being C₃-C₁₀ cycloalkyl,C₆-C₁₀ aryl, 5 to 10-membered heteroaryl, or 4 to 10-memberedheterocycloalkyl, and T₃ being optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,thiol, carboxyl, cyano, nitro, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ alkoxyl, C₁-C₆ haloalkyl, C₁-C₆ haloalkoxyl, C₁-C₆alkylthio, C₁-C₆ alkylsulfonyl, C₁-C₆ haloalkylsulfonyl, C₁-C₆alkylcarbonyl, C₁-C₆ alkoxycarbonyl, oxo, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₄-C₁₂ alkylcycloalkyl, C₆-C₁₀aryl, C₆-C₁₀ aryloxyl, C₇-C₁₄ alkylaryl, C₆-C₁₀ aminoaryloxyl, C₆-C₁₀arylthio, 4 to 6-membered heterocycloalkyl optionally substituted withhalo, C₁-C₄ alkyl, C₁-C₄ haloalkyl, 5 to 6-membered heteroaryloptionally substituted with halo, C₁-C₄ alkyl, and C₁-C₆ alkyl that issubstituted with hydroxy, halo, C₁-C₆ alkoxycarbonyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-memberedheteroaryl optionally further substituted with halo, hydroxyl, or C₁-C₆alkoxyl;

m is 1 or 2; and

n is 1 or 2.

Compounds of formula IV, methods of making thereof, and methods of usethereof can be found in WO 2012/075492, which is incorporated herein byreference.

In certain embodiments, at least one of m and n is 2.

In certain embodiments, each of G and J independently is OR_(a). Incertain embodiments, each of G and J is OH.

In certain embodiments, L₁ is N(Y). In certain embodiments, L, is SO orSO₂.

In certain embodiments, Y is R_(d). In certain embodiments, R_(d) isC₁-C₆ alkyl.

In certain embodiments, L₂ is absent.

In certain embodiments, R₉ is

In certain embodiments, R⁹ is

In certain embodiments, R_(i) is H or C₁-C₆ alkyl (e.g., methyl, ethyl,n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl andn-hexyl).

In certain embodiments, at least one of R_(e), R_(f), R_(g), and R_(h)is halo (such as F, CI, and Br), C₁-C₆ alkoxyl optionally substitutedwith one or more halo (such as OCH₃, OCH₂CH₃, O-iPr, and OCF₃), C₁-C₆alkylsulfonyl optionally substituted with one or more halo (such asSO₂CF₃), or C₁-C₆ alkyl optionally substituted with one or more halo(such as CH₃, i-propyl, n-butyl, and CF₃). In certain embodiments, atleast one of R_(e), R_(f), R_(g), and R_(h) is selected from the groupconsisting of F, Cl, CF₃, OCF₃, C₁-C₄ alkyl, and C₁-C₄ alkoxyl.

In certain embodiments,

is unsubstituted benzimidazolyl or one of the following groups:

In certain embodiments, R₉ is

In certain embodiments, R⁹ is hydrogen.

In certain embodiments, D is O. In certain embodiments, D is NRj. Incertain embodiments, R_(j) is H. In certain embodiments, D isCR_(j)R_(k). In certain embodiments, each of R_(j) and R_(k) is H.

In certain embodiments, E is -M₃-T₃, in which M₃ is a bond or C₁-C₃alkyl linker, T₃ is phenyl, naphthyl, thienyl, cyclopropyl, orcyclohexyl, and T₃ is optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,thiol, carboxyl, cyano, nitro, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ alkoxyl, C₁-C₆ haloalkyl, C₁-C₆ haloalkoxyl, C₁-C₆alkylthio, C₁-C₆ alkylsulfonyl, C₁-C₆ alkylcarbonyl, C₁-C₆alkoxycarbonyl, oxo, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino,C₃-C₈ cycloalkyl, C₄-C₁₂ alkylcycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryloxyl,C₇-C₁₄ alkylaryl, C₆-C₁₀ aminoaryloxyl, C₆-C₁₀ arylthio, 4 to 6-memberedheterocycloalkyl optionally substituted with C₁-C₄ alkyl, 5 to6-membered heteroaryl optionally substituted with C₁-C₄ alkyl, and C₁-C₆alkyl that is substituted with hydroxy, C₁-C₆ alkoxycarbonyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, or 5 to6-membered heteroaryl. In certain embodiments, T₃ is phenyl optionallysubstituted with one or more substituents selected from the groupconsisting of halo, hydroxyl, carboxyl, cyano, nitro, C₁-C₆ alkyl (e.g.,methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl,s-pentyl and n-hexyl), C₁-C₆ alkoxyl, C₁-C₆ haloalkyl, C₁-C₆haloalkoxyl, C₁-C₆ alkylsulfonyl, C₆-C₁₀ aryl (e.g., phenyl ornaphthyl), and C₆-C₁₀ aryloxyl, and C₇-C₁₄ alkylaryl. In certainembodiments, E is

In certain embodiments, X is N. In certain embodiments, X is CR_(X). Incertain embodiments, X is CH.

In certain embodiments, Q is NH₂ or NHR_(b), in which R_(b) is -M₁-T₁,M₁ being a bond or C₁-C₆ alkyl linker and T₁ being C₃-C₈ cycloalkyl. Incertain embodiments, Q is H.

In certain embodiments, R₁, R₂, R⁴, R₅, R⁶, R₇, and R₈ are each H.

In certain embodiments, when R⁸ is halo and is attached to the samecarbon atom as J, then J is not hydroxyl. In certain embodiments, whenR₈ is halo and is attached to the same carbon atom as G, then G is nothydroxyl. In certain embodiments, T₂ is not halo when M₂ is SO₂, SO, S,CO or O.

In certain embodiments, T₂ is a 4-8 membered heterocycloalkyl which isbound to M₂ via a heteroatom. In certain embodiments, T₂ is a 4-8membered heterocycloalkyl which is bound to M₂ via a N atom. In certainembodiments, T₂ is a 4-8 membered heterocycloalkyl which is bound to M₂via a C atom.

In certain embodiments, the compound is of formula III-a:

or a pharmaceutically acceptable salt or ester thereof, wherein G, J, Q,X, L₁, L₂, R₁, R₂, R₄, R₅, R₆ R⁷, R₈, R₉, m, and n are as describedherein for compounds of formula IV.

In certain embodiments, the compound is of the formula IV-b or IV-c:

pharmaceutically acceptable salt or ester thereof, wherein G, J, Q, X,L₁, L₂, R₁, R₂, R⁴, R₅, R₆, R₇, R₈, R_(e), R_(f), R_(g), R_(h), R_(i), mand n are as described herein for compounds of formula IV. In certainembodiments, the compound is of formula IV-d:

pharmaceutically acceptable salt or ester thereof, wherein G, J, Q, X,L₁, L₂, R₁, R₂, R₄, R₅, R₆, R₇, R₈, D, E, m, and n are as describedherein for formula IV.

Exemplary compound useful in methods described include:

or pharmaceutically acceptable salts thereof.

In some embodiments, Dot1L is inhibited by contacting the differentiatedcell with a composition comprising a compound of formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments, Dot1L is inhibited by contacting the differentiatedcell with a composition comprising a compound of formula:

As used herein, “alkyl”, “C₁, C₂, C₃, C₄, C₅ or C₆ alkyl” or “C₁-C₆alkyl” is intended to include C₁, C₂, C₃, C₄, C₅ or C₆ straight chain(linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆branched saturated aliphatic hydrocarbon groups. For example, C₁-C₆alkyl is intended to include C₁, C₂, C₃, C₄, C₅ and C₆ alkyl groups.Examples of alkyl include moieties having from one to six carbon atoms,such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl. In certain embodiments,a straight chain or branched alkyl has six or fewer carbon atoms (e.g.,C₁-C₆ for straight chain, C₃-C₆ for branched chain), and in anotherembodiment, a straight chain or branched alkyl has four or fewer carbonatoms.

As used herein, the term “cycloalkyl” refers to a saturated orunsaturated nonaromatic hydrocarbon mono- or multi-ring system having 3to 30 carbon atoms (e.g., C₃-C₁₀). Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, andadamantyl. The term “heterocycloalkyl” refers to a saturated orunsaturated nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic,or 11-14 membered tricyclic ring system having one or more heteroatoms(such as O, N, S, or Se). Examples of heterocycloalkyl groups include,but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, and tetrahydrofuranyl.

The term “optionally substituted alkyl” refers to unsubstituted alkyl oralkyl having designated substituents replacing one or more hydrogenatoms on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

An “arylalkyl” or an “aralkyl” moiety is an alkyl substituted with anaryl (e.g., phenylmethyl (benzyl)). An “alkylaryl” moiety is an arylsubstituted with an alkyl (e.g., methylphenyl).

As used herein, “alkyl linker” is intended to include C₁, C₂, C₃, C₄, C₅or C₆ straight chain (linear) saturated divalent aliphatic hydrocarbongroups and C₃, C₄, C₅ or C₆ branched saturated aliphatic hydrocarbongroups. For example, C₁-C₆ alkyl linker is intended to include C₁, C₂,C₃, C₄, C₅ and C₆ alkyl linker groups. Examples of alkyl linker include,moieties having from one to six carbon atoms, such as, but not limitedto, methyl (—CH₂—), ethyl (—CH₂CH₂—), n-propyl (—CH₂CH₂CH₂—), i-propyl(—CHCH₃CH₂—), n-butyl (—CH₂CH₂CH₂CH₂—), s-butyl (—CHCH₃CH₂CH₂—), i-butyl(—C(CH₃)₂CH₂—), n-pentyl (—CH₂CH₂CH₂CH₂CH₂—), s-pentyl(—CHCH₃CH₂CH₂CH₂—) or n-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₂—).

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. For example, the term “alkenyl” includes straightchain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenylgroups. In certain embodiments, a straight chain or branched alkenylgroup has six or fewer carbon atoms in its backbone (e.g., C₂-C₆ forstraight chain, C₃-C₆ for branched chain). The term “C₂-C₆” includesalkenyl groups containing two to six carbon atoms. The term “C₃-C₆”includes alkenyl groups containing three to six carbon atoms.

The term “optionally substituted alkenyl” refers to unsubstitutedalkenyl or alkenyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl;sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. For example, “alkynyl” includes straight chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. Incertain embodiments, a straight chain or branched alkynyl group has sixor fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). The term “C₂-C₆” includes alkynyl groupscontaining two to six carbon atoms. The term “C₃-C₆” includes alkynylgroups containing three to six carbon atoms.

The term “optionally substituted alkynyl” refers to unsubstitutedalkynyl or alkynyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Other optionally substituted moieties (such as optionally substitutedcycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both theunsubstituted moieties and the moieties having one or more of thedesignated substituents.

“Aryl” includes groups with aromaticity, including “conjugated,” ormulticyclic systems with at least one aromatic ring and do not containany heteroatom in the ring structure. Examples include phenyl, benzyl,1,2,3,4-tetrahydronaphthalenyl, etc.

“Heteroaryl” groups are aryl groups, as defined above, except havingfrom one to four heteroatoms in the ring structure, and may also bereferred to as “aryl heterocycles” or “hetero aromatic s.” As usedherein, the term “heteroaryl” is intended to include a stable 5- or6-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclicaromatic heterocyclic ring which consists of carbon atoms and one ormore heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6heteroatoms, or e.g. 1, 2, 3, 4, 5, or 6 heteroatoms, independentlyselected from the group consisting of nitrogen, oxygen and sulfur. Thenitrogen atom may be substituted or unsubstituted (i.e., N or NR whereinR is H or other substituents, as defined). The nitrogen and sulfurheteroatoms may optionally be oxidized (i.e., N→O and S(O)_(p), wherep=1 or 2). It is to be noted that total number of S and O atoms in thearomatic heterocycle is not more than 1. Examples of heteroaryl groupsinclude pyrrole, furan, thiophene, thiazole, isothiazole, imidazole,triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine,pyridazine, pyrimidine, and the like.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine,indolizine.

In the case of multicyclic aromatic rings, only one of the rings needsto be aromatic (e.g., 2,3-dihydroindole), although all of the rings maybe aromatic (e.g., quinoline). The second ring can also be fused orbridged.

The aryl or heteroaryl aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,phosphate, phosphonato, phosphinato, amino (including alkylamino,dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor hetero aromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings, which are not aromatic so as to form amulticyclic system (e.g., tetralin, methylenedioxyphenyl).

As used herein, “carbocycle” or “carbocyclic ring” is intended toinclude any stable monocyclic, bicyclic or tricyclic ring having thespecified number of carbons, any of which may be saturated, unsaturated,or aromatic. For example, a C₃-C₁₄ carbocycle is intended to include amonocyclic, bicyclic or tricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14 carbon atoms. Examples of carbocycles include, but arenot limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl,cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl,adamantyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl,naphthyl, indanyl, adamantyl and tetrahydronaphthyl. Bridged rings arealso included in the definition of carbocycle, including, for example,[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane and[2.2.2]bicyclooctane. A bridged ring occurs when one or more carbonatoms link two non-adjacent carbon atoms. In one embodiment, bridgerings are one or two carbon atoms. It is noted that a bridge alwaysconverts a monocyclic ring into a tricyclic ring. When a ring isbridged, the substituents recited for the ring may also be present onthe bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) and spiro ringsare also included.

As used herein, “heterocycle” includes any ring structure (saturated orpartially unsaturated) which contains at least one ring heteroatom(e.g., N, O or S). Examples of heterocycles include, but are not limitedto, morpholine, pyrrolidine, tetrahydrothiophene, piperidine, piperazineand tetrahydrofuran. Examples of heterocyclic groups include, but arenot limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl,benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl,carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl,furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl,oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.

The term “substituted,” as used herein, means that any one or morehydrogen atoms on the designated atom is replaced with a selection fromthe indicated groups, provided that the designated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.When a substituent is oxo or keto (i.e., ═O), then 2 hydrogen atoms onthe atom are replaced. Keto substituents are not present on aromaticmoieties. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stablecompound” and “stable structure” are meant to indicate a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchformula.

Combinations of substituents and/or variables are permissible, but onlyif such combinations result in stable compounds.

When any variable (e.g., R*) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with 0-2R* moieties, thenthe group may optionally be substituted with up to two R* moieties andR* at each occurrence is selected independently from the definition ofR. Also, combinations of substituents and/or variables are permissible,but only if such combinations result in stable compounds.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O″.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo andiodo. The term “perhalogenated” generally refers to a moiety wherein allhydrogen atoms are replaced by halogen atoms. The term “haloalkyl” or“haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or morehalogen atoms.

The term “carbonyl” includes compounds and moieties which contain acarbon connected with a double bond to an oxygen atom. Examples ofmoieties containing a carbonyl include, but are not limited to,aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “carboxyl” refers to —COOH or its C₁-C₆ alkyl ester.

“Acyl” includes moieties that contain the acyl radical (R—C(O)—) or acarbonyl group. “Substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by, for example, alkyl groups,alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Aroyl” includes moieties with an aryl or heteroaromatic moiety bound toa carbonyl group. Examples of aroyl groups include phenylcarboxy,naphthyl carboxy, etc.

“Alkoxyalkyl,” “alkylaminoalkyl,” and “thioalkoxyalkyl” include alkylgroups, as described above, wherein oxygen, nitrogen, or sulfur atomsreplace one or more hydrocarbon backbone carbon atoms.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups or alkoxyl radicals include, but are notlimited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxygroups. Examples of substituted alkoxy groups include halogenated alkoxygroups. The alkoxy groups can be substituted with groups such asalkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

The term “ether” or “alkoxy” includes compounds or moieties whichcontain an oxygen bonded to two carbon atoms or heteroatoms. Forexample, the term includes “alkoxyalkyl,” which refers to an alkyl,alkenyl, or alkynyl group covalently bonded to an oxygen atom which iscovalently bonded to an alkyl group.

The term “ester” includes compounds or moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc.

The term “thioalkyl” includes compounds or moieties which contain analkyl group connected with a sulfur atom. The thioalkyl groups can besubstituted with groups such as alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes moieties which contain a sulfur atombonded to two carbon atoms or heteroatoms. Examples of thioethersinclude, but are not limited to alkthioalkyls, alkthioalkenyls, andalkthioalkynyls. The term “alkthioalkyls” include moieties with analkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bondedto an alkyl group. Similarly, the term “alkthioalkenyls” refers tomoieties wherein an alkyl, alkenyl or alkynyl group is bonded to asulfur atom which is covalently bonded to an alkenyl group; andalkthioalkynyls” refers to moieties wherein an alkyl, alkenyl or alkynylgroup is bonded to a sulfur atom which is covalently bonded to analkynyl group.

As used herein, “amine” or “amino” refers to unsubstituted orsubstituted —NH₂. “Alkylamino” includes groups of compounds whereinnitrogen of —NH₂ is bound to at least one alkyl group. Examples ofalkylamino groups include benzylamino, methylamino, ethylamino,phenethylamino, etc. “Dialkylamino” includes groups wherein the nitrogenof —NH₂ is bound to at least two additional alkyl groups. Examples ofdialkylamino groups include, but are not limited to, dimethylamino anddiethylamino. “Arylamino” and “diarylamino” include groups wherein thenitrogen is bound to at least one or two aryl groups, respectively.“Aminoaryl” and “aminoaryloxy” refer to aryl and aryloxy substitutedwith amino. “Alkylarylamino,” “alkylaminoaryl” or “arylaminoalkyl”refers to an amino group which is bound to at least one alkyl group andat least one aryl group.

“Alkaminoalkyl” refers to an alkyl, alkenyl, or alkynyl group bound to anitrogen atom which is also bound to an alkyl group. “Acylamino”includes groups wherein nitrogen is bound to an acyl group. Examples ofacylamino include, but are not limited to, alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “amide” or “aminocarboxy” includes compounds or moieties thatcontain a nitrogen atom that is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups thatinclude alkyl, alkenyl or alkynyl groups bound to an amino group whichis bound to the carbon of a carbonyl or thiocarbonyl group. It alsoincludes “arylaminocarboxy” groups that include aryl or heteroarylmoieties bound to an amino group that is bound to the carbon of acarbonyl or thiocarbonyl group. The terms “alkylaminocarboxy”,“alkenylaminocarboxy”, “alkynylaminocarboxy” and “arylaminocarboxy”include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties,respectively, are bound to a nitrogen atom which is in turn bound to thecarbon of a carbonyl group. Amides can be substituted with substituentssuch as straight chain alkyl, branched alkyl, cycloalkyl, aryl,heteroaryl or heterocycle. Substituents on amide groups may be furthersubstituted.

Compounds of the present invention that contain nitrogens can beconverted to N-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to affordother compounds of the present invention. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O″). Furthermore, inother instances, the nitrogens in the compounds of the present inventioncan be converted to N-hydroxy or N-alkoxy compounds. For example,N-hydroxy compounds can be prepared by oxidation of the parent amine byan oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent invention includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like. In addition, a crystal polymorphism may bepresent for the compounds represented by the formula. It is noted thatany crystal form, crystal form mixture, or anhydride or hydrate thereofis included in the scope of the present invention. Furthermore,so-called metabolite which is produced by degradation of the presentcompound in vivo is included in the scope of the present invention.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

A carbon atom bonded to four nonidentical substituents is termed a“chiral center.” “Chiral isomer” means a compound with at least onechiral center. Compounds with more than one chiral center may existeither as an individual diastereomer or as a mixture of diastereomers,termed “diastereomeric mixture.” When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cyclobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the compounds of the present invention maybe depicted as different chiral isomers or geometric isomers. It shouldalso be understood that when compounds have chiral isomeric or geometricisomeric forms, all isomeric forms are intended to be included in thescope of the present invention, and the naming of the compounds does notexclude any isomeric forms.

Furthermore, the structures and other compounds discussed in thisinvention include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), amine-enamine andenamine-enamine. Benzimidazoles also exhibit tautomerism, when thebenzimidazole contains one or more substituents in the 4, 5, 6 or 7positions, the possibility of different isomers arises. For example,2,5-dimethyl-1H-benzo[d]imidazole can exist in equilibrium with itsisomer 2,6-dimethyl-1H-benzo[d] imidazole via tautomerization.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofthe compounds does not exclude any tautomer form.

The term “crystal polymorphs”, “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different X-ray diffraction patterns, infrared spectral, meltingpoints, density hardness, crystal shape, optical and electricalproperties, stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

Compounds of the invention may be crystalline, semi-crystalline,non-crystalline, amorphous, mesomorphous, etc.

Compounds described herein include the compounds themselves, as well astheir N-oxides, salts, their solvates, and their prodrugs, ifapplicable. A salt, for example, can be formed between an anion and apositively charged group (e.g., amino) on a substituted purine or7-deazapurine compound. Suitable anions include chloride, bromide,iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate,malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate,lactate, naphthalenesulfonate, and acetate.

Likewise, a salt can also be formed between a cation and a negativelycharged group (e.g., carboxylate) on a substituted purine or7-deazapurine compound. Suitable cations include sodium ion, potassiumion, magnesium ion, calcium ion, and an ammonium cation such astetramethylammonium ion. The substituted purine or 7-deazapurinecompounds also include those salts containing quaternary nitrogen atoms.Examples of prodrugs include esters and other pharmaceuticallyacceptable derivatives, which, upon administration to a subject, arecapable of providing active substituted purine or 7-deazapurinecompounds.

Additionally, the compounds described herein, for example, the salts ofthe compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include hemihydrates, monohydrates, dihydrates,trihydrates, etc. Nonlimiting examples of solvates include ethanolsolvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O. A hemihydrate isformed by the combination of one molecule of water with more than onemolecule of the substance in which the water retains its molecular stateas H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds represented byFormula (I) are substituted purine compounds or substituted7-deazapurine compounds, and have Formula (I) as a common core.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides, tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176,1996.

All isotopes of atoms occurring in the compounds described herein areintended to be encompassed. Isotopes include those atoms having the sameatomic number but different mass numbers. By way of general example andwithout limitation, isotopes of hydrogen include tritium and deuterium,and isotopes of carbon include C-13 and C-14.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference, in particular for the teaching that isreferenced hereinabove.

EXAMPLES Example 1 Materials and Methods:

shRNA Cloning

shRNAs were designed using the RNAi Codex26. 97-mer oligos (Table 2)These were amplified with the following primer pair (SEQ ID NOs: 1 and2)

Forward: GATGGCTGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCGReverse: GTCTAGAGGAATTCCGAGGCAGTAGGC.

PCR products were gel purified, digested with EcoRI and XHoI and ligatedinto the MSCV-PM (Openbiosystems) vector. Clones were verified bysequencing. shRNA targeting the firefly luciferase was used as acontrol27. Nanog shRNA was previously described28.

Production of Viral Supernatants

293T cells were plated at a density of 2.5×106 cells per 10-cm dish. Thenext day, cells were transfected with 2 μg viral vector, 2 μg Gag-Polvector and 0.22 μg VSV-G plasmid using 20 μl Fugene 6 (Roche AppliedScience #1181509001) in 400 μl DMEM per plate. Supernatant was collected48 h and 72 h post-transfection and filtered through 45 μm pore sizefilters. For concentration, viral supernatants were mixed with PEG3350solution (Sigma P3640, dissolved in PBS, 10% final concentration) andleft overnight at 4 C. The next day, supernatants were centrifuged at2500 rpm for 20 minutes, and the pellets were re-suspended in PBS.Titering was performed on 293 Ts. For shRNA infections, 500 ul of viralsupernatant was used to infect 25,000 cells in the presence of 10 ug/mlprotamine sulfate. For fluorescent labeling of dh1fs, we usedlentiviruses PRRL-GFP (Addgene #12252) and FUdGW-Tomato (Addgene#22771).

Reprogramming Assays

dH1f cells were first infected with shRNA viruses at high MOI to ensureall cells received at least one vector (Gauged by puromycin resistanceof parallel infected wells). 25,000 shRNA-infected dH1f cells were thenplated per well in 12-well plates and infected overnight with eitherretroviral (MOI 2.5)7 or lentiviral (Addgene #21162, 21164; 100-200 μlsupernatant)29 reprogramming factors. For 2-factor reprogramming, Oct4and Sox2 viruses were used at an MOI of 5. 6 days later, cells weretrypsinized and re-plated 1:4 to 1:6 onto 6-well plates. Medium waschanged to hES medium daily until Day 21 when plates were fixed. Smallmolecule inhibitor of Dot1L, EPZ004777 (a gift from Epizyme, Inc.,Cambridge, Mass.) was dissolved in DMSO as a 10 mM stock and was addedat the indicated concentrations. For Dot1L rescue experiments, anMSCV-based retroviral vector encoding human Dot1L with or withoutmutations in the SAM binding site (gifts of Y. Zhang) were mutagenizedat the shRNA target site using QuikChange II XL Site-DirectedMutagenesis Kit (Agilent Technologies). In certain experiments, Nanogand Lin28 expression was achieved using lentivirus (Addgene #21163).IMR-90 and MRC5 human diploid fibroblasts were purchased from ATCC and50000 cells were used in reprogramming experiments. Statistical analysiswas performed using a Student's t-test.

Microarray Analysis

Total RNA was extracted from three independent culture plates for eachconditions with an RNeasy Mini kit (Qiagen). Synthesis of cRNA fromtotal RNA and hybridization/scanning of microarrays were performed withAffymetrix GeneChip products (HGU133A) as described in the GeneChipmanual. Normalization of the raw gene expression data, quality controlchecks, and subsequent analyses were done with the open-source R-projectstatistical software (R Development Core Team,2007)(http://www.r-project.org/) together with Bioconductor packages.Raw data files (.CEL) were converted into probe set values by RMAnormalization. Genes were selected at a threshold of Log Ratio 0.4. Themicroarray data have been deposited in National Center for BiotechnologyInformation Gene Expression Omnibus (GEO) and are accessible through GEOSeries accession number GSE29253.

SYBR-Green Real-Time RT-PCR

Total RNA was extracted using RNeasy Mini kit coupled with RNase-freeDNase set (Qiagen) and reverse transcribed with Hexanucleotide Mix(Roche). The resulting cDNAs were used for PCR using SYBR-Green MasterPCR mix (Applied Biosystem) in triplicates. All quantitations werenormalized to an endogenous Beta-Actin control. The relativequantitation value for each target gene compared to the calibrator forthat target is expressed as 2-(Ct-Cc) (Ct and Cc are the mean thresholdcycle differences after normalizing to Beta-Actin). List of primers canbe found in Table 3.

Immunostaining

Immunostaining of reprogramming plates were performed as described⁸.Briefly, cells were fixed with 4% p-formaldehyde and stained withbiotin-anti-Tra-1-60 (eBioscience, #13-8863-82, 1:250) and streptavidinhorseradish peroxidase (Biolegend, #405210, 1:500) diluted in PBS (3%),FCS (0.3%) Triton X-100. Staining was developed with the Vector labs DABkit (#SK-4100), and iPSC colonies quantified with ImageJ software. Forthe characterization of shDot1l-iPS cells, single colonies were put ontoMEF coated 96-well plates. The plates were fixed for 20 min with 4%p-formaldehyde/PBS (+/+), washed several times with PBS (+/+) andincubated overnight at 4° C. with primary antibody and Hoechst dilutedin 3% donkey serum/3% BSA Fraction VII/0.01% Triton X-100/PBS (+/+);Hoechst, Invitrogen #H3570 (1:20,000), Tra-1-81/A488 (BD #560174),SSEA-4/A647 (BD #560219), Tra-1-60/A647 (BD #560122), Nanog, rabbitpolyclonal (Abcam #ab21624), Oct4, rabbit polyclonal (Abcam #ab19857).For Nanog and Oct4, donkey anti-rabbit IgG/A555 (Molecular Probes#A31572) secondary antibody was used. After several washes with PBS(+/+), images were acquired using a BD Pathway 435 imager equipped witha x 10 objective.

Teratoma Formation Assay

iPSCs grown on MEFs were harvested with Collagenease IV (1 mg/ml inDMEM/F12). Cell clumps from one 6-well plate were resuspended in 50 μlDMEM/F12, 100 μl collagen I (Invitrogen-#A1064401) and 150 μlhESC-qualified matrigel (BD Biosciences-#354277). Cell clumps were theninjected into the hind limb femoral muscles (100 μl suspension per leg)of Rag2 γ/c mice. After 6-8 weeks, teratomas were harvested and fixed inBouin's solution overnight. Samples were then embedded in paraffin, andsections were stained with hematoxylin/eosin (Rodent HistopathologyCore, Harvard Medical School, Boston, Mass., USA).

Characterization of iPS Cells

Embryoid body differentiation was performed as described (Loewer). Tocheck for the presence of the reprogramming transgenes, genomic DNA wasisolated using DNeasy Blood & Tissue Kit (Qiagen) and PCR was performedwith specific primers to the endogenous or the viral trangenes³.

ChIP Assays

ChIP-seq was performed as described with slight modifications²⁵. 300 000cells were fixed at room temperature in PBS 1% formalin (v/v) for 10minutes with gentle agitations. Fixation was stopped by the addition ofglycine (125 mM final concentration) and agitation for 5 min at roomtemperature. Fixed cells were washed twice in ice-cold PBS, resuspendedin 100 ul of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH8.1). Chromatin was sheared by sonication to about 100-500 bp fragmentsusing bioruptor (diagenode, Denville, N.J.) and diluted tenfold withdilution buffer (0.01% SDS, 1.1% Triton-X100, 1.2 mM EDTA, 16.7 mMTris-HCl, pH 8.1, 167 mM NaCl). Antibodies against specific histonemodifications was added to sonicated chromatin solution and incubated at4 degree overnight with gentle agitation. The antibodies used wereanti-H3K27me3 (Millipore 07-449) and anti-H3K79me2 (abcam 3594). Immunecomplexes were collected by incubation with 20 ul of Protein A/G agarosebeads (Millipore) for an hour at 4 degree with gentle agitation.Precipitates were washed sequentially with ice cold low salt wash (0.1%SDS, 1% Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl, pH8.1, 150 mM NaCl),high salt wash (0.1% SDS, 1% Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl, pH8.1, 500 mM NaCl), LiCl wash (0.25M LiCl, 1% IGEPAL CA-630, 1%deoxycholic acid, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and TE wash (1 mMEDTA, 10 mM Tris-HCl, pH 8.1) for 5 mins each at 4 degree with gentleagitation. Samples were centrifuged briefly in between washes to collectthe beads. Immunoprecipitated DNA was eluted by incubating beads with150 ul elution buffer (l % SDS, 0.1 M NaHCO3) with gentle agitation for15 mins at room temperature. Elution was repeated once and eluates werecombined, sodium chloride (final concentration of 0.2M) were added tothe eluate and eluates were incubated at 65 degree overnight to reversecrosslinking. DNA was purified using PCR purification spin column(Qiagen). For ChIP sequencing, ChIP DNA libraries were made followingIllumina ChIP-seq library preparation kit and subjected to Solexasequencing (Illumina) at Center for Cancer Computational Biology, DanaFaber Cancer Institute.

TABLE 2 shRNA Sequences evaluated HairpinTGCTGTTGACAGTGAGCGAGCGATTGCTCCAGGAATTTAATAGTGAAGCCACAGATGTATTAAATTCCTGGAGCAATCGCCTGCCTACTGCCTCGGA (SEQ ID NO: 3)TGCTGTTGACAGTGAGCGCCGTGCCCATTCCCTGTGTCAATAGTGAAGCCACAGATGTATTGACACAGGGAATGGGCACGTTGCCTACTGCCTCGGA (SEQ ID NO: 4)TGCTGTTGACAGTGAGCGAGGTGATGACTTCAGTCTCTACTAGTGAAGCCACAGATGTAGTAGAGACTGAAGTCATCACCCTGCCTACTGCCTCGGA (SEQ ID NO: 5)TGCTGTTGACAGTGAGCGCGGATGGAGAGGTGTACTGCATTAGTGAAGCCACAGATGTAATGCAGTACACCTCTCCATCCTTGCCTACTGCCTCGGA (SEQ ID NO: 6)TGCTGTTGACAGTGAGCGACGATGCCAGCAGTCATGCAAATAGTGAAGCCACAGATGTATTTGCATGACTGCTGGCATCGCTGCCTACTGCCTCGGA (SEQ ID NO: 7)TGCTGTTGACAGTGAGCGAGCAGCAACGGATACATCTTAATAGTGAAGCCACAGATGTATTAAGATGTATCCGTTGCTGCCTGCCTACTGCCTCGGA (SEQ ID NO: 8)TGCTGTTGACAGTGAGCGACGATGCCAGCAGTCATGCAAATAGTGAAGCCACAGATGTATTTGCATGACTGCTGGCATCGCTGCCTACTGCCTCGGA (SEQ ID NO: 9)TGCTGTTGACAGTGAGCGAGGGATTCAGATGTCACCTTAATAGTGAAGCCACAGATGTATTAAGGTGACATCTGAATCCCGTGCCTACTGCCTCGGA (SEQ ID NO: 10)TGCTGTTGACAGTGAGCGCCGAGTGTTATATTTGTGAATATAGTGAAGCCACAGATGTATATICACAAATATAACACTCGTTGCCTACTGCCTCGGA (SEQ ID NO: 11)TGCTGTTGACAGTGAGCGCGCACCTCTGAACTTCAGAATATAGTGAAGCCACAGATGTATATTCTGAAGTTCAGAGGTGCATGCCTACTGCCTCGGA (SEQ ID NO: 12)TGCTGTTGACAGTGAGCGAGGACGGGAGCTCCACTGTGAATAGTGAAGCCACAGATGTATTCACAGTGGAGCTCCCGTCCGTGCCTACTGCCTCGGA (SEQ ID NO: 13)TGCTGTTGACAGTGAGCGCGGAGCTCACCTTTGATTACAATAGTGAAGCCACAGATGTATTGTAATCAAAGGTGAGCTCCTTGCCTACTGCCTCGGA (SEQ ID NO: 14)TGCTGTTGACAGTGAGCGACCTCGGTATCTCTAAGAGGAATAGTGAAGCCACAGATGTATTCCTCTTAGAGATACCGAGGGTGCCTACTGCCTCGGA (SEQ ID NO: 15)TGCTGTTGACAGTGAGCGACGGGCCTTCGTGTACATCAATTAGTGAAGCCACAGATGTAATTGATGTACACGAAGGCCCGCTGCCTACTGCCTCGGA (SEQ ID NO: 16)TGCTGTTGACAGTGAGCGCGCCCGTTACTGCTTCAGCAATTAGTGAAGCCACAGATGTAATTGCTGAAGCAGTAACGGGCATGCCTACTGCCTCGGA (SEQ ID NO: 17)TGCTGTTGACAGTGAGCGCGCTTAGTATATGTGTACTTAATAGTGAAGCCACAGATGTATTAAGTACACATATACTAAGCTTGCCTACTGCCTCGGA (SEQ ID NO: 18)TGCTGTTGACAGTGAGCGACCAAATCTTCAGGTGTTCAATTAGTGAAGCCACAGATGTAATTGAACACCTGAAGATTTGGGTGCCTACTGCCTCGGA (SEQ ID NO: 19)TGCTGTTGACAGTGAGCGCCCTGATAGTCAGCATGCGAATTAGTGAAGCCACAGATGTAATTCGCATGCTGACTATCAGGTTGCCTACTGCCTCGGA (SEQ ID NO: 20)TGCTGTTGACAGTGAGCGCGGGCTTTCATGTTATCTATAATAGTGAAGCCACAGATGTATTATAGATAACATGAAAGCCCATGCCTACTGCCTCGGA (SEQ ID NO: 21)TGCTGTTGACAGTGAGCGACCTGAAGAGTCCAATGATGATTAGTGAAGCCACAGATGTAATCATCATTGGACTCTTCAGGGTGCCTACTGCCTCGGA (SEQ ID NO: 22)TGCTGTTGACAGTGAGCGCAGGATTCTGGCCAAACAGAAATAGTGAAGCCACAGATGTATTTCTGTTTGGCCAGAATCCTTTGCCTACTGCCTCGGA (SEQ ID NO: 23)TGCTGTTGACAGTGAGCGACGGAGGGCCAAGCACTATAAATAGTGAAGCCACAGATGTATTTATAGTGCTTGGCCCTCCGGTGCCTACTGCCTCGGA (SEQ ID NO: 24)TGCTGTTGACAGTGAGCGAACCGGTTAAGAGATTCTTATTTAGTGAAGCCACAGATGTAAATAAGAATCTCTTAACCGGTCTGCCTACTGCCTCGGA (SEQ ID NO: 25)TGCTGTTGACAGTGAGCGCGGCATTATGCTTGTTGTACAATAGTGAAGCCACAGATGTATTGTACAACAAGCATAATGCCATGCCTACTGCCTCGGA (SEQ ID NO: 26)TGCTGTTGACAGTGAGCGACCATTGTAAGTGTTGTTTCTATAGTGAAGCCACAGATGTATAGAAACAACACTTACAATGGGTGCCTACTGCCTCGGA (SEQ ID NO: 27)TGCTGTTGACAGTGAGCGAGGAAAGAATATGCATAGAATATAGTGAAGCCACAGATGTATATTCTATGCATATTCTTTCCGTGCCTACTGCCTCGGA (SEQ ID NO: 28)TGCTGTTGACAGTGAGCGCCGGAACTCAACCATTAAGCAATAGTGAAGCCACAGATGTATTGCTTAATGGTTGAGTTCCGTTGCCTACTGCCTCGGA (SEQ ID NO: 29)TGCTGTTGACAGTGAGCGCGGGACTGCAATTATTCAGTATTAGTGAAGCCACAGATGTAATACTGAATAATTGCAGTCCCTTGCCTACTGCCTCGGA (SEQ ID NO: 30)TGCTGTTGACAGTGAGCGACCAGTGGCCAGTTCACTGTATTAGTGAAGCCACAGATGTAATACAGTGAACTGGCCACTGGCTGCCTACTGCCTCGGA (SEQ ID NO: 31)TGCTGTTGACAGTGAGCGAGCAGTTACATGCATACTTCAATAGTGAAGCCACAGATGTATTGAAGTATGCATGTAACTGCCTGCCTACTGCCTCGGA (SEQ ID NO: 32)TGCTGTTGACAGTGAGCGCGCTCTGTAATCTCGTTTCAAATAGTGAAGCCACAGATGTATTTGAAACGAGATTACAGAGCATGCCTACTGCCTCGGA (SEQ ID NO: 33)TGCTGTTGACAGTGAGCGCCCTCCTGATTATTCAGAATATTAGTGAAGCCACAGATGTAATATTCTGAATAATCAGGAGGTTGCCTACTGCCTCGGA (SEQ ID NO: 34)TGCTGTTGACAGTGAGCGACGAAGAGCTCTTCTTTGATTATAGTGAAGCCACAGATGTATAATCAAAGAAGAGCTCTTCGCTGCCTACTGCCTCGGA (SEQ ID NO: 35)TGCTGTTGACAGTGAGCGCGCCAGTAACAAGAAAGAGAAATAGTGAAGCCACAGATGTATTTCTCTTTCTTGTTACTGGCATGCCTACTGCCTCGGA (SEQ ID NO: 36)TGCTGTTGACAGTGAGCGACCTGCATCATGACTCAGAATTTAGTGAAGCCACAGATGTAAATTCTGAGTCATGATGCAGGGTGCCTACTGCCTCGGA (SEQ ID NO: 37)TGCTGTTGACAGTGAGCGCCAACATTATGGGCATCGAGAATAGTGAAGCCACAGATGTATTCTCGATGCCCATAATGTTGTTGCCTACTGCCTCGGA (SEQ ID NO: 38)TGCTGTTGACAGTGAGCGACGAGCTACAAAGCATGGGAAATAGTGAAGCCACAGATGTATTTCCCATGCTTTGTAGCTCGGTGCCTACTGCCTCGGA (SEQ ID NO: 39)TGCTGTTGACAGTGAGCGACGTCCGCAGGAACTTAACTTATAGTGAAGCCACAGATGTATAAGTTAAGTTCCTGCGGACGCTGCCTACTGCCTCGGA (SEQ ID NO: 40)TGCTGTTGACAGTGAGCGCCCTGAGGATAACTCAATATAATAGTGAAGCCACAGATGTATTATATTGAGTTATCCTCAGGTMCCTACTGCCTCGGA (SEQ ID NO: 41)TGCTGTTGACAGTGAGCGCCCGGGAACAGAGAATGTTTAATAGTGAAGCCACAGATGTATTAAACATTCTCTGTTCCCGGTTGCCTACTGCCTCGGA (SEQ ID NO: 42)TGCTGTTGACAGTGAGCGCGGTCTCAGGCGCCAGTGGAAATAGTGAAGCCACAGATGTATTTCCACTGGCGCCTGAGACCATGCCTACTGCCTCGGA (SEQ ID NO: 43)TGCTGTTGACAGTGAGCGCGCCTAGTAAATTACAGAAGAATAGTGAAGCCACAGATGTATTCTTCTGTAATTTACTAGGCATGCCTACTGCCTCGGA (SEQ ID NO: 44)TGCTGTTGACAGTGAGCGCGCTTCTAGGCAGAGTTGCTTATAGTGAAGCCACAGATGTATAAGCAACTCTGCCTAGAAGCTTGCCTACTGCCTCGGA (SEQ ID NO: 45)TGCTGTTGACAGTGAGCGACGCATATATTTGCAGTATGAATAGTGAAGCCACAGATGTATTCATACTGCAAATATATGCGCTGCCTACTGCCTCGGA (SEQ ID NO: 46)TGCTGTTGACAGTGAGCGACCGTCCCGTGGAGTCGCTAAATAGTGAAGCCACAGATGTATTTAGCGACTCCACGGGACGGGTGCCTACTGCCTCGGA (SEQ ID NO: 47)TGCTGTTGACAGTGAGCGCGCCCTCCCTGTCCTTTCCAGATAGTGAAGCCACAGATGTATCTGGAAAGGACAGGGAGGGCTTGCCTACTGCCTCGGA (SEQ ID NO: 48)TGCTGTTGACAGTGAGCGCCGCCAGCCTTCGCTTCTGAAATAGTGAAGCCACAGATGTATTTCAGAAGCGAAGGCTGGCGTTGCCTACTGCCTCGGA (SEQ ID NO: 49)TGCTGTTGACAGTGAGCGCGAGCTTCATGGGATTGGTAAATAGTGAAGCCACAGATGTATTTACCAATCCCATGAAGCTCATGCCTACTGCCTCGGA (SEQ ID NO: 50)TGCTGTTGACAGTGAGCGAACCTTTCCAGCCATAGAGATTTAGTGAAGCCACAGATGTAAATCTCTATGGCTGGAAAGGTGTGCCTACTGCCTCGGA (SEQ ID NO: 51)TGCTGTrGACAGTGAGCGCGCTTTCAAGCTCATCTGTTATTAGTGAAGCCACAGATGTAATAACAGATGAGCTTGAAAGCTTGCCTACTGCCTCGGA (SEQ ID NO: 52)TGCTGTTGACAGTGAGCGAACAGTTGGATTCTTTAGAGAATAGTGAAGCCACAGATGTATTCTCTAAAGAATCCAACTGTCTGCCTACTGCCTCGGA (SEQ ID NO: 53)TGCTGTTGACAGTGAGCGACGAGAGAGTTAGCTGACTTTATAGTGAAGCCACAGATGTATAAAGTCAGCTAACTCTCTCGGTGCCTACTGCCTCGGA (SEQ ID NO: 54)TGCTGTTGACAGTGAGCGACCTGATTATATCCAGTAACACTAGTGAAGCCACAGATGTAGTGTTACTGGATATAATCAGGGTGCCTACTGCCTCGGA (SEQ ID NO: 55)TGCTGTTGACAGTGAGCGCGCCCAAGGTCAAGGAGATTATTAGTGAAGCCACAGATGTAATAATCTCCTTGACCTTGGGCTTGCCTACTGCCTCGGA (SEQ ID NO: 56)TGCTGTTGACAGTGAGCGCGGCATCCACTGTGAATGATAATAGTGAAGCCACAGATGTATTATCATTCACAGTGGATGCCATGCCTACTGCCTCGGA (SEQ ID NO: 57)TGCTGTTGACAGTGAGCGCGCTGTCTCTCTTTGATGGAATTAGTGAAGCCACAGATGTAATTCCATCAAAGAGAGACAGCATGCCTACTGCCTCGGA (SEQ ID NO: 58)TGCTGTTGACAGTGAGCGCGCCTGCAAGGACATGGTTAAATAGTGAAGCCACAGATGTATTTAACCATGTCCTTGCAGGCTTGCCTACTGCCTCGGA (SEQ ID NO: 59)TGCTGTTGACAGTGAGCGACGCACCTACTCCAAGTTCAAATAGTGAAGCCACAGATGTATTTGAACTTGGAGTAGGTGCGCTGCCTACTGCCTCGGA (SEQ ID NO: 60)TGCTGTTGACAGTGAGCGCCGAGTCTGGCTTTGAGAGTTATAGTGAAGCCACAGATGTATAACTCTCAAAGCCAGACTCGTTGCCTACTGCCTCGGA (SEQ ID NO: 61)TGCTGTTGACAGTGAGCGAGCCATGGAAATGCTATCAATGTAGTGAAGCCACAGATGTACATTGATAGCATTTCCATGGCCTGCCTACTGCCTCGGA (SEQ ID NO: 62)TGCTGTTGACAGTGAGCGCAGATGGAAGATGATATAGATATAGTGAAGCCACAGATGTATATCTATATCATCTTCCATCTTTGCCTACTGCCTCGGA (SEQ ID NO: 63)TGCTGTTGACAGTGAGCGCCCAAATCTTCTCCTGTCAGTATAGTGAAGCCACAGATGTATACTGACAGGAGAAGATTTGGATGCCTACTGCCTCGGA (SEQ ID NO: 64)TGCTGTTGACAGTGAGCGAAGAGATTATTTCTCAAGATGATAGTGAAGCCACAGATGTATCATCTTGAGAAATAATCTCTCTGCCTACTGCCTCGGA (SEQ ID NO: 65)TGCTGTTGACAGTGAGCGCAGAGGGAAAGTGTATGATAAATAGTGAAGCCACAGATGTATTTATCATACACTTTCCCTCTTTGCCTACTGCCTCGGA (SEQ ID NO: 66)TGCTGTTGACAGTGAGCGCGGAAAGAACGGAAATCTTAAATAGTGAAGCCACAGATGTATTTAAGATTTCCGTTCTTTCCATGCCTACTGCCTCGGA (SEQ ID NO: 67)TGCTGTTGACAGTGAGCGCGCAGTTATGCTCTIAATGCTTTAGTGAAGCCACAGATGTAAAGCATTAAGAGCATAACTGCTTGCCTACTGCCTCGGA (SEQ ID NO: 68)TGCTGTTGACAGTGAGCGCGCATGCATGACTTTAATCTTATAGTGAAGCCACAGATGTATAAGATTAAAGTCATGCATGCTTGCCTACTGCCTCGGA (SEQ ID NO: 69)TGCTGTTGACAGTGAGCGAAACATGTGTAAGCTGCGGCCCTAGTGAAGCCACAGATGTAGGGCCGCAGCTTACACATGTTCTGCCTACTGCCTCGGA (SEQ ID NO: 70)TGCTGTTGACAGTGAGCGAAAGGATGTGGTCCGAGTGTGGTAGTGAAGCCACAGATGTACCACACTCGGACCACATCCTTCTGCCTACTGCCTCGGA (SEQ ID NO: 71)

TABLE 3 qRT PCR primers used qRT-PCR SEQ SEQ primers Forward ID IDReverse ActB TGAAGTGTGACGTGGACATC 72 73 GGAGGAGCAATGATCTTGAT DNMT1GAATCTCTTGCACGAATTTCTGC 74 75 CATGAGCACCGTTCTCCAAGG DNMT3ACCGATGCTGGGGACAAGAAT 76 77 CCCGTCATCCACCAAGACAC EedGCGGAGGAATATGTCCGAGAG 78 79 AGAGGTCTGGATTGCTGTTCT ezh2ATGGGCCAGACTGGGAAGAA 80 81 TGGAAAATCCAAGTCACTGGTC suz12AGTTGTCCAATAAGGCAAGTTCC 82 83 ACGAGTCACTCTAAATAGCAACG hG9aCATTTCCGCATGAGTGATGATGT 84 85 CAGGCCACCTCCTGAGTTC MBD1TTACCCCAGGTGAAGCAAGAG 86 87 CCAATACGGGAGAAGTCAGGAC MBD2CCCACAACGAATGAATGAACAGC 88 89 TGAAGACCTTTGGGTAGTTCCA MBD3CTGGGAGAGGGAAGAAGTGC 90 91 CGGAAGTCGAAGGTGCTCAG MeCP2AGCAGAGACATCAGAAGGGTC 92 93 CGGCCAGATTTCCTTTGCTT Dot1LGCTGCCGGTCTACGATAAACA 94 95 AGCTTGAGATCCGGGATTTCT Suv39h1ATATCCAGACTCAGAGAGCACC 96 97 CAGCTCCCTTTCTAAGTCCTTG Setdb1TGGATGACAAAAGATGTGAGTGG 98 99 CCATATTTGGACGTGTCCTGAG smyd2GCCCTACAGTAAGCACTATCCT 100 101 AGTCTCCCTAGCTTCAACCAC bmi1CCACCTGATGTGTGTGCTTTG 102 103 TTCAGTAGTGGTCTGGTCTTGT ring1TCAGAACTCATGTGCCCTATCT 104 105 GCAGGTAGGACACTCCTTGT yy1AGAAGAGCGGCAAGAAGAGTT 106 107 CAACCACTGTCTCATGGTCAATA ezh1TCAATGAAGCCTGTGAGTGGA 108 109 CAATGCAACTGTGTTCAGTGAC Nr2f1CAGGCCAGTACGCACTCAC 110 111 TGTTCTCGATGCCCATAATGTTG MBD4CCCCACCGTCACCTCTAGT 112 113 GTAGCACCAAACTGAGCAGAA suv39h2GGCTAAACAAAGGATAGCTCTGC 114 115 TGAAAGAAGCAATCTGTGCATGA ehmt1CAACGCCGTAGACAGCGAG 116 117 CTCCCCGTCCTTATTGTCGAG RunX1CCCTAGGGGATGTTCCAGAT 118 119 TGAAGCTTTTCCCTCTTCCA AFPAGCTTGGTGGTGGATGAAAC 120 121 CCCTCTTCAGCAAAGCAGAC GATA4CTAGACCGTGGGTTTTGCAT 122 123 TGGGTTAAGTGCCCCTGTAG BrachuryACCCAGTTCATAGCGGTGAC 124 125 CAATTGTCATGGGATTGCAG NcamATGGAAACTCTATTAAAGTGAACCTG 126 127 TAGACCTCATACTCAGCATTCCAGT

Example 2: Screening for Inhibitors and Enhancers of Reprogramming

To examine the influence of modifiers on somatic cell reprogramming, aloss-of-function approach was employed to interrogate the role of 22select genes in DNA and histone methylation pathways. FIG. 1 shows anon-limiting outline of a screen for inhibitors and enhancers ofreprogramming. FIG. 1A illustrates a protocol for screening shRNA poolsin iPS cell generation.

In one example, a pool of 3 hairpins was tested for each of 22 targetgenes and knockdown efficiencies were observed of >60% for 21 out of 22targets by qRT-PCR (FIG. 6). FIG. 6 shows the measurement of knockdownefficiency of the indicated shRNA pools in dH1f cells measured byquantitative reverse transcription PCR (qRT-PCR). Expression values foreach gene were normalized to those measured in control shRNAfibroblasts. Human fibroblasts were used as a system in which to studyreprogramming. Human fibroblasts were differentiated from H1 humanembryonic stem cells (dH1fs), which have a higher reprogrammingefficiency than primary adult dermal fibroblasts, thus yielding areproducible baseline^(7,8). dH1fs were infected with shRNA pools (athigh multiplicity of infection to ensure all cells received an shRNAvector) followed by super-infection with reprogramming vectorsexpressing Oct4, Sox2, Klf4 and c-Myc (OSKM), and identified theresulting iPSCs by Tra-1-60 staining (FIG. 1A).

Eight shRNA pools negatively affected reprogramming (FIG. 1B). FIG. 1Bshows the number of Tra-1-60+ colonies, as quantified by ImageJ, 21 daysafter OSKM transduction of 25,000 dH1f cells previously infected withpools of shRNAs against the indicated genes (3 vectors per gene).Corresponding images of representative Tra-1-60-stained reprogrammingwells are shown in the lower panel. The dotted lines indicate 3 standarddeviations from the mean number of colonies detected in control shRNAwells. Among the target genes were Pou5F1 (Oct4, included as a control),and Ehmt1 and SetDB1, two H3K9 methyltransferases whose histone mark wasassociated with transcriptional repression. The remaining five shRNApools all targeted components of polycomb repressive complexes (PRC),which are major mediators of gene silencing and heterochromatinformation. Inhibition of PRC1 (Bmi1, Ring1) and PRC2 components (Ezh2,Eed, Suz12) did not have a significant effect on fibroblastproliferation but resulted in significantly fewer iPSC colonies than thecontrol shRNA (FIG. 1C, FIG. 7). FIG. 1C shows the validation of primaryscreen hits that decrease reprogramming efficiency. Quantification ofTra-1-60+ iPSC colonies expressed as fold-change relative to controlshRNA. Data correspond to the average and s.e.m. *P<0.05, **P<0.01compared to control shRNA-expressing fibroblasts. RepresentativeTra-1-60-stained reprogramming wells are shown in the lower panel. FIG.7 shows cell proliferation upon PRC1/2 knockdown. Depicted are relativecell growth rates of fibroblasts infected with the indicated shRNAvectors targeting PRC1 and PRC2 components. Relative control shRNAinfected fibroblasts 5 days after shRNA transduction (n=3; error bars,±s.e.m).

This is of particular significance given the recent finding that PRC2component Ezh2 is necessary for fusion-based reprogramming⁹. Thus,interference with chromatin-modifying enzymes that mediate repressivechromatin domains reduces reprogramming and is consistent with theimportance of gene silencing of the somatic cell program duringgeneration of iPSC.

Example 3: Suppression of Dot1L Expression Enhances ReprogrammingEfficiency and Substitutes for Klf4 and Myc

In contrast to genes whose functions appear to be required forreprogramming, inhibition of three genes enhanced reprogramming:YY1,Suv39H1, and Dot1L (FIG. 1D). FIG. 1D shows the validation of primaryscreen hits that increase reprogramming efficiency. Quantification ofTra-1-60+ iPSC colonies expressed as fold-change relative to controlshRNA. Data correspond to the average and s.e.m. *P<0.05, **P<0.01compared to control shRNA-expressing fibroblasts. RepresentativeTra-1-60-stained reprogramming wells are shown in the lower panel. YY1is a transcription factor that activates or represses transcription in acontext-dependent manner^(10,11), whereas Suv39H1 is a histone H3K9methyltransferase implicated in heterochromatin formation¹². Enzymesthat modify H3K9 were associated with both inhibition and enhancement ofreprogramming in this study, which suggested that unraveling the precisemechanisms for their effects might be challenging. The studies focusedon Dot1L, a histone H3 Lysine 79 methyltransferase whose role inreprogramming has not been previously studied¹³. Three Dot1L-targetinghairpin vectors were evaluated independently for knockdown efficiencyand utilized the two shRNAs that resulted in the most significantdownregulation of Dot1L and concomitant decrease in global H3K79 levels(FIG. 8). FIG. 8 shows Dot1L mRNA and total H3K79me2 levels underknock-down conditions. FIG. 8A shows the knock-down efficiency ofindividual hairpins targeting Dot1L as measured by qRT-PCR. FIG. 8Bshows the total H3K79me2 levels in control and shDot1L infectedfibroblasts assessed by western blotting 6 days after shRNAtransduction.

Following infection with OSKM, fibroblasts expressing Dot1L shRNA formedsignificantly more iPSC colonies than control fibroblasts, and thedegree of enhancement in reprogramming correlated with the degree ofDot1L knockdown (FIG. 2A, FIG. 7A). In addition, the enhancedreprogramming phenotype elicited by Dot1L knockdown could be reversed byoverexpressing an shRNA-resistant wildtype Dot1L, but not by acatalytically-inactive Dot1L, indicating that inhibition of catalyticactivity of Dot1L is key to reprogramming¹⁴ (FIG. 2A). FIG. 2 shows thatDot1L inhibition enhances reprogramming efficiency and substitutes forKlf4 and Myc. FIG. 2A shows the fold change in the reprogrammingefficiency of dH1f cells infected with 2 independent Dot1L shRNAs orco-infected with shRNA-2 and a vector expressing an shRNA-resistantwild-type or catalytically dead mutant Dot1L. Data correspond to theaverage and s.e.m.; n-independent experiments. *P<0.01 controlshRNA-expressing fibroblasts.

To further document the competitive advantage of Dot1L shRNA-expressingfibroblasts (shDot1L) during reprogramming, experiments were performedin which fibroblasts infected with Dot1L shRNA or a control vector weredifferentially labeled with either tdTomato or GFP and co-cultured priorto superinfection with OSKM (FIG. 9).

FIG. 9 shows same-well reprogramming of admixed control and Dot1Linhibited cell populations. Control and shDot1L dH1f cells were infectedwith either a GFP- or tdTomato-expressing lentivirus, and mixed in a 1:1ratio prior to OSKM infection. The middle panel shows a quantificationof morphologically discernible iPS-like colonies that were scored on thebasis of the fluorescent protein expression. Data correspond to the meanratio of GFP+ colonies to Tomato+ colonies in each co-mixture and s.d.(n=2). Lower panels show representative images of either GFP or tdTomatoexpressing iPSC colonies (arrowheads) derived from the indicatedco-mixed cell populations.

The emerging iPSC colonies were then scored for tdTomato or GFPfluorescence, which indicated their cell of origin. Co-mixture oftdTomato- and GFP-labeled control cells resulted in a 1:1 ratio of redand green iPSC colonies indicating that labeling per se did not affectrelative reprogramming efficiency. In contrast, wells containingco-mixtures of tdTomato-shDot1L and GFP-shCntrl cells generated 3-foldmore red colonies than green colonies, indicating that Dot1L-inhibitedcells reprogram more efficiently under identical conditions (FIG. 9).The experiments were repeated with additional strains of humanfibroblasts, and a 3-fold and 6-fold increases in reprogrammingefficiency for IMR-90 and MRC-5 cells was observed, respectively,indicating that findings with dH1fs were broadly applicable to otherhuman fibroblast strains (FIG. 2B). FIG. 2B shows the number ofTra-1-60+ colonies derived from 50,000 control and Dot1LshRNA-expressing IMR-90 and MRC5 human diploid fibroblasts. Datacorrespond to the average and s.d. *P<0.05 compared to controlshRNA-expressing fibroblasts (n=2). Lower panels show representativeTra-1-60-stained wells from the indicated conditions. iPS cellsgenerated through Dot1L inhibition exhibited a morphology characteristicof embryonic stem cells and stained positively for SSEA4, SSEA3,Tra-1-81, Oct4 and Nanog (FIG. 2C). FIG. 2C shows immunohistochemistryexpression analysis of pluripotency markers, SSEA4, SSEA3, Oct4, Nanogand Tra-1-81 in expanded shDot1L-iPS colonies derived from 4-factor(OSKM) and 2-factor (OS) reprogramming.

Additionally, these cells were able to differentiate into all threeembryonic germ layers in vitro and in teratomas (FIG. 2D and FIG. 10A),indicating that iPSCs generated following Dot1L inhibition display allof the hallmarks of pluripotency. FIG. 2D shows hematoxylin and eosinstaining of representative OS-, OSM-, OSKM-shDot1L-iPS teratomasexhibiting ectoderm (neural rosettes), mesoderm (cartilage), andendoderm (gut-like endothelium) differentiation. FIG. 10 shows acharacterization of shDot1L iPS cells. FIG. 10A shows the downregulationof endogenous OCT4 mRNA, as well as the upregulation of differentiationmarkers GATA4 and AFP (endoderm), RUNX1 and Brachury (mesoderm), andNCAM (ectoderm) in day 8 EBs derived from shCntrl-OSKM, shDot1L-OSKM,and shDot1L-OS iPS cells as judged by qRT-pCR. Expression values arerepresented relative to undifferentiated controls.

To assess whether Dot1L inhibition could replace any of the 4 exogenousreprogramming factors, shDot1L and shCntrl fibroblasts were infectedwith 3 factors, omitting one factor at a time. In the absence of Oct4 orSox2 no iPSC colonies emerged in both types of fibroblasts. When eitherKlf4 or Myc was omitted, shDot1L fibroblasts were able to give rise torobust numbers of Tra-1-60 positive colonies while control cells yieldedvery few colonies as reported previously³. It was examined whether Dot1Lsuppression would suffice to reprogram fibroblasts in the absence ofboth Klf4 and c-Myc. Indeed, shDot1L fibroblasts infected with only Oct4and Sox2 gave rise to Tra-1-60-positive colonies, whereas controlfibroblasts did not (FIG. 2E). FIG. 2E shows Tra-1-60 staining of wholeplates of shCntrl and shDot1L fibroblasts 21 days after reprogramming inthe absence of each factor or both Klf4 and c-Myc. Two-factor iPSCsderived by Dot1L inhibition exhibited a typical ES cell morphology andhad silenced GFP expression from the exogenous reprogramming vectors(FIG. 10B). FIG. 10B shows the morphology changes and retroviralsilencing in colonies emerging in 4-factor (OSKM) and 2-factor (OS)reprogramming of control and Dot1L-inhibited cells. Green fluorescenceindicates persistent GFP expression derived from the pMIG reprogrammingvectors. Note that after 2-factor (OS) transduction, colonies arisingfrom only shDot1L or iDot1L-treated fibroblasts silence the transgenesas indicated by the absence of GFP expression, whereas shCntrlfibroblasts yield transformed cell clusters that retain factorexpression. PCR on genomic DNA isolated from expanded colonies indicatedthe presence of Oct4 and Sox2, but not the Klf4 and c-Myc transgenes(FIG. 10C). FIG. 10C shows PCR-based detection of transgenes in thegenomic DNA of 2- and 3-factor iPSC lines derived from shDot1L cellswith primers designed to amplify either endogenous loci or thevirally-encoded transgenes. H9ES and dH1f cells served as negativecontrols for the transgenes and vector plasmids and OKSM-derived iPSClines were used as positive controls.

The OS-shDot1L-iPS cells had all of the hallmarks of pluripotency asgauged by endogenous pluripotency factor expression and the ability toform all three embryonic germ layers in vitro and in teratomas (FIG. 2C,2D and FIG. 10A). These findings indicate that Dot1L inhibition canreprogram cells to pluripotency in the presence of only Oct4 and Sox2.

It was examined whether the cellular mechanisms by which Dot1Linhibition promotes reprogramming. It was observed that in establishedhuman iPS clones derived from shDot1L fibroblasts, Dot1L inhibition wasno longer evident, reflecting the known silencing of retroviruses thatoccurs during reprogramming (FIG. 11A).

FIG. 11 shows Dot1L knockdown dynamics during reprogramming. FIG. 11Ashows Dot1L mRNA levels in iPSCs derived from shCntrl and shDot1L iPSCsrelative to levels in the starting control dh1F cells as measured byqRT-PCR. FIG. 11B shows Dot1L mRNA levels in shDot1L cells measured byqRT-PCR every 3 days after OSKM expression normalized to levels observedin control cells prior to OSKM expression. FIG. 11C shows Dot1L mRNAlevels in control cells measured by qRT-PCR every 3 days after OSKMexpression normalized to levels observed in control cells prior to OSKMexpression.

Examination of Dot1L knockdown levels during the course of reprogrammingrevealed that silencing occurred by day 15 after OSKM transduction(FIGS. 11B, C). Since Dot1L inhibition seemed to work in the initialphases of reprogramming, it was examined if the proliferation rates ofshDot1L and shCntrl cells before and after infection with the OSKMreprogramming factors and found them to be comparable (FIGS. 12A and12B). FIG. 12 shows the growth dynamics of shDot1L cells pre- andpost-OSKM transduction. FIG. 12A shows the cumulative populationdoubling rates of shCntrl and shDot1L cells over a period of 14 daysprior to reprogramming (n=3; error bars, ±s.e.m). FIG. 12B shows therelative cell growth rates of shCntrl and shDot1L cells prior to and 6days after OSKM transduction (n-3; error bars, ±s.e.m).

There was also no difference in the number of senescent cells, as gaugedby senescence-associated β-gal staining following OSKM infection (FIG.12C, data not shown). In addition, the level of retroviral reprogrammingfactor expression was similar between control and Dot1L-inhibited cells(FIG. 12C). FIG. 12C shows qRT-PCR quantification of viral transcriptlevels using transgene-specific primers in control or iDot1L treateddH1f cells 3 days after infection with OSM or OS expressingretroviruses.

Previous studies indicated that Dot1L null cells have increasedapoptosis and accumulation of cells in G2 phase¹³. However, asignificant increase in apoptosis or change in the cell cycle profile ofDot1L knock-down fibroblasts was not observed (FIG. 13). FIG. 13 showsapoptosis and a cell cycle profile of Dot1L-inhibited cells. FIG. 13Ashows the percentage of apoptotic cells in the indicated cellpopulations 5 days after treatment (iDot1L-10 um) or shRNA infectionmeasured by PI/Annexin staining (n=3; error bars, ±s.d.). FIG. 13B showsa cell cycle profile of untreated or iDot1L (10 uM for 5 days) treatedfibroblasts as measured by PI staining.

It was also examined whether Dot1L knockdown, in addition to increasingreprogramming efficiency, accelerated the kinetics of reprogramming.FIG. 14 shows the kinetics of iPSC colony formation upon Dot1Lknockdown. FIG. 14A shows the number of Tra-1-60+ iPSC coloniesgenerated by shCntrl and shDot1L dH1f cells on Day 14 and Day 21 ofreprogramming (n=3; error bars, ±s.e.m). FIG. 14B shows the number ofTra-1-60+ cell clusters per field as judged by live-staining and imagingon Day 10 after OSKM transduction (n=12 fields, ±s.e.m).Immunofluorescence analysis during reprogramming revealed significantlygreater numbers of Tra-1-60-positive cell clusters on day 10 (FIG. 14A)and recognizable Tra-1-60-positive colonies on days 14 and 21 in shDot1Lcultures (FIG. 14B), indicating that the emergence of iPSC colonies isaccelerated upon Dot1L inhibition. FIG. 12C shows qRT-PCR quantificationof viral transcript levels using transgene-specific primers in controlor iDot1L treated dH1f cells 3 days after infection with OSM or OSexpressing retroviruses.

When the reprogramming experiments were extended by 10 more days (atwhich time the already-formed iPSC colonies begin to differentiate),shDot1L cells still yielded more iPSC colonies than controls (FIG. 14C).FIG. 14C shows a modified schema for testing the reprogrammingefficiency of 10,000 OSKM infected dH1f cells without replating ontoMEFs and a longer incubation period of 30 days. The graph on the rightshows the number of Tra-1-60+ iPSC colonies generated through thismodified protocol (n=2; error bars, ±s.e.m) When the reprogrammingexperiments were extended by 10 more days (at which time thealready-formed iPSC colonies begin to differentiate), shDot1L cellsstill yielded more iPSC colonies than controls (FIG. 14C). Takentogether, Dot1L inhibition both accelerates the emergence of iPSCs andincreases the total yield of the reprogramming process.

Example 4: Small Molecule Inhibitor of Dot1L Enhances Reprogramming andReplaces Klf4 and Myc

In addition to the shRNA mediated knockdown of Dot1L, a small moleculeinhibitor of Dot1L catalytic activity was used to further validate thefindings¹⁵. This inhibitor (EPZ004777, referred to as iDot1L and shownbelow) abrogated H3K79 methylation robustly at 1 uM to 10 uMconcentration range and led to 3-4 fold enhancement of reprogramming ofhuman fibroblasts (FIG. 3A, FIG. 15).

FIG. 3 shows that the small molecule inhibitor of Dot1L increasesefficiency and substitutes for Klf4 and Myc. FIG. 3A shows the foldchange in the reprogramming efficiency of dH1f cells treated with iDot1Lat the indicated concentrations for 21 days. Data correspond to theaverage and s.d.; n=3. *P<0.001 compared untreated fibroblasts. FIG. 15shows that the small molecule inhibitor of Dot1L increases reprogrammingefficiency. FIG. 15A shows total H3K79me2 levels in fibroblasts treatedwith EPZ004777 (iDot1L) at the indicated concentrations for 5 days asassessed by western blotting. FIG. 15B shows representative images ofcontrol or iDot1L treated reprogramming plates stained with Tra-1-60 onday 21.

Knockdown of Dot1L protein in combination with inhibitor treatment didnot result in a further increase of reprogramming efficiency therebyreinforcing the previous observation that inhibition of catalyticactivity of Dot1L is key to reprogramming (FIG. 3B). FIG. 3B shows thenumber of Tra-1-60+ colonies 21 days after OSKM transduction of 25,000control fibroblasts, iDot1L treated fibroblasts (10 uM) and iDot1Ltreated fibroblasts expressing the Dot1L shRNA. Data correspond toaverage and s.d (n=2). Representative Tra-1-60-stained reprogrammingwells are in the lower panel. Similar to shRNA mediated suppression ofDot1L, chemical inhibition of Dot1L allowed reprogramming in the absenceof either Klf4 or Myc and was able to replace both of these exogenousfactors during reprogramming (FIG. 3C). FIG. 3C shows Tra-1-60 stainingof whole plates of untreated or iDot1L(10 uM) fibroblasts 21 days afterreprogramming in the absence of Klf4, c-Myc or both. Thus, it waspossible to generate two-factor iPSCs robustly and reproducibly eitherby shRNA mediated suppression of Dot1L or chemical inhibition of itsmethyl transferase activity.

It was assessed whether these observations can be extended to the murinesystem. It was observed that, similar to human fibroblasts, iDot1Ltreatment led to 3-fold enhancement of reprogramming of Oct4-GFP MEFs(FIG. 3D). FIG. 3D shows the number of AP+ colonies derived from OSKMtransduced untreated or iDot1L treated (10 um) Oct4-GFP Mefs. Datacorrespond to the average and s.d.; n=4. *P<0.001 compared untreatedMEFs. Representative AP-stained wells and GFP-positive iPS coloniesderived from each condition are shown in the lower panel.

The colonies resulting from Dot1L inhibition were GFP-positiveindicating the activation of the endogenous Oct4 gene. Furthermore,reprogramming of tail-tip fibroblasts (TTFs) derived from a conditionalknockout Dot1L mouse strain yielded significantly more iPS colonies uponCre mediated excision of Dot1L (FIG. 16A). FIG. 16 shows thereprogramming of Dot1L conditional knockout tail-tip fibroblasts TTFs.FIG. 16A shows an example of fold change in Alkaline Phosphatasepositive (AP+) colonies upon OSKM transduction of TTFs derived fromDot1L fl/fl mice. TTFs were first infected with a MSCV-CRE-ER vector.Vehicle(Ethanol) or 4-OHT was added to cultures at the same time as OSKMinfection. Data correspond to average and s.e.m. (n-6). The completeexcision of both floxed Dot1L alleles in iPSC clones derived fromhomozygous TTFs was confirmed by genomic PCR (FIG. 16B). FIG. 16B showsgenomic PCR to detect wildtype, floxed or deleted Dot1L alleles in iPScolonies derived from reprogramming of the indicated startingCre-expressing TTFs in the presence or absence of 4OHT. Dot1L inhibitionalso increased reprogramming efficiency of inducible iPS-derived“secondary” MEFs (FIG. 16C). FIG. 16C shows the number of AP+ coloniesupon doxycycline addition to iPS-derived secondary MEFs in the presenceof iDot1L (10 um). Data correspond to average and s.d (n=2).

Taken together these results demonstrate that Dot1L inhibition enhancesreprogramming of mouse cells as well.

To further dissect out the crucial time window for Dot1L inhibition,human fibroblasts undergoing reprogramming were treated with iDot1L at 1week intervals. It was observed that Dot1L inhibition either in thefirst or the second week was sufficient to enhance reprogramming whereaspretreatment for 5 days prior to OSKM transduction had no effect (FIG.3E). FIG. 3E shows the number of Tra-1-60+ colonies 21 days after OSKMtransduction of 25,000 untreated fibroblasts, and fibroblasts treatedwith iDot1L(10 uM) for the indicated time periods during reprogramming.Data correspond to average and s.d (n=3). RepresentativeTra-1-60-stained reprogramming wells are shown in the upper panel.These. findings indicate that Dot1L inhibition at early to middle stagesin the reprogramming process facilitates the acquisition ofpluripotency.

Example 5: Dot1L Inhibition During Reprogramming Induces Nanog and Lin28Expression

Since the effects of Dot1L inhibition were evident early in thereprogramming process, it was investigate if gene expression changes inDot1L-inhibited cells soon after transduction with reprogramming factorscould reveal insights into the molecular mechanisms involved. A globalgene-expression analyses on control and shDot1L fibroblasts prior to and6 days after OSKM transduction was performed along with cells that weretreated with iDot1L. Although thousands of genes were induced orrepressed upon OSKM expression, relatively few genes were differentiallyexpressed in shDot1L cells on Day 6 of reprogramming (22 up, 23 down;).At this time point, inhibitor treated cells exhibited broader geneexpression changes (405 up and 175 down), presumably due to morecomplete inhibition of K79me2 levels.

To understand the mechanism by which Dot1L inhibition substitutes forKlf4, gene expression analyses on control and shDot1L cells wereperformed upon 3-factor infection with OSM. While 94 genes weredifferentially upregulated in shDot1L cells in the absence of Klf4, theintersection of this set of genes with the set differentiallyupregulated in 4-factor reprogramming of shDot1L and inhibitor treatedcells yielded only 5 common genes (FIG. 4A, 4B). These five genes wereLefty1, Lin28A, Lum, Upp1 and Nanog. Nanog and Lin28 were upregulated inall three instances of Dot1L inhibition. These two genes are part of thecore pluripotency network of human ES cells^(16,17,18) and havepreviously been shown to be sufficient for reprogramming humanfibroblasts into iPSC when used in combination with Oct4 and Sox2⁴.

FIG. 4 shows that Nanog and Lin28 are important for the enhancement ofreprogramming by Dot1L inhibition. FIG. 4A shows the overlap ofdifferentially upregulated genes in shDot1L cells 6 Days post-OSKM andOSM transduction with the genes upregulated in OSKM transducediDotL-treated cells. FIG. 4B shows heat maps showing differentialexpression levels of commonly upregulated genes in OSKM transducedDot1L-inhibited cells.

The possibility was explored that Nanog and Lin28 upregulation wasresponsible for the enhanced reprogramming observed following Dot1Linhibition and validated their upregulation in shDot1L fibroblastscompared to control fibroblasts 6 days after transduction withreprogramming factors (FIG. 4C). FIG. 4C shows the expression levels ofNanog, Lin28, Rex1, and Dnmt3b in shDot1L cells 6 days post-OSM or -OStransduction relative to shCntrl cells as measured by qRT-PCR.Interestingly at this early time-point, no upregulation of Rex1 andDnmt3b was observed, two other well-characterized pluripotency genes,suggesting that Dot1L inhibition does not broadly upregulate thepluripotency network. Suppression of either Nanog or Lin28 expressionusing lentiviral shRNAs abrogated the 2-factor reprogramming of shDot1Lfibroblasts, indicating the essential roles of Nanog and Lin28 in thisprocess (FIG. 4D, FIG. 17B). FIG. 4D shows the number of Tra-1-60+ iPSCcolonies upon knockdown of Nanog or Lin28 in 2-factor reprogramming ofshDot1L cells (n=2; error bars, +s.e.m). FIG. 17 shows an example of theknockdown efficiency of Nanog and Lin28 during 2-factor reprogramming.FIG. 17A shows Lin28 and Nanog mRNA levels in shCntrl, shDot1L oriDot1L-treated cells prior to reprogramming. Expression values arerepresented relative to HIES cells. FIG. 17B shows Lin28 and Nanog mRNAlevels in shDot1L cells expressing Lin28 shRNA or Nanog shRNA 6-daysafter OS transduction. Expression values are represented relative toshDot1L cells prior to OS infection.

It was hypothesized that if Nanog and Lin28 upregulation is the majormechanism by which Dot1L inhibition enhances reprogramming, theninclusion of Nanog and Lin28 in the OSKM reprogramming cocktail wouldnot confer any additional enhancement to shDot1L cells. In fact, nosignificant difference in the number of colonies generated between4-factor (OSKM) and 6-factor (OSKMNL) reprogramming of shDot1L cells wasobserved, although the colonies were larger in the latter conditions(FIG. 4E, FIG. 17C). FIG. 4E shows the fold-change in Tra-1-60+ iPSCcolonies in 4-factor (OSKM) and 6-factor (OSKM+Nanog+Lin28)reprogramming of shCntrl and shDot1L fibroblasts relative to control4-factor reprogramming of shCntrl cells. Plates were stained on Day 20of reprogramming. Corresponding images of representative Tra-1-60stained reprogramming wells are shown above (n=2; error bars, ±s.e.m).FIG. 17C shows the average Tra-1-60 positive colony size in 4-factor(OSKM) and 6-factor (OSKM+Nanog+Lin28) reprogramming of shCntrl andshDot1L fibroblasts relative to control 4-factor reprogramming ofshCntrl cells as shown in FIG. 4C. In control fibroblasts, Nanog andLin28 expression did enhance 4-factor reprogramming by 2.4 fold, andthus significantly phenocopied Dot1L inhibition (FIG. 4E). Takentogether, these data indicate that Dot1L inhibition requires the actionof both Nanog and Lin28 to substitute for Klf4 and c-Myc and enhancereprogramming.

Example 6: Genome-Wide Analysis of H3K79Me2 Marks During Reprogramming

To gain insight into the genome-wide chromatin changes that arefacilitated by Dot1L inhibition during reprogramming, ChIP-seq forH3K79me2 and H3K27me3 in human ES cells as well as fibroblastsundergoing reprogramming with or without the iDot1L treatment wereperformed (FIG. 18). FIG. 18 shows a Chip-seq experimental design. FIG.18A shows dH1f fibroblasts that were pre-treated with iDot1L for 5 daysat 10 uM (Day 0) and then infected with OSKM. 6 days later, cells wereharvested for the ChIP (Day 6). FIG. 18B shows the number of raw andaligned reads from Illumina sequencing for each ChIP sample. In both EScells and fibroblasts, the presence H3K27me3 and H3K79me2 was mutuallyexclusive (FIG. 19). FIG. 19 shows the relationship between H3K79me2 andH3K27me3. FIG. 19A shows a genome-wide representation of the relationbetween H3K79me2 and H3K27me3 in ES cells. FIG. 19B shows a genome-widerepresentation of the relation between H3K79me2 and H3K27me3 inFibroblasts. Genes marked by H3K79me2 specifically in ES cells werepluripotency factors, their downstream targets and genes involved inepithelial cell adhesion such as CDH1 (165 genes, FIG. 20). In contrast,genes marked by H3K79me2 specifically in fibroblasts were significantlyenriched in gene sets associated with epithelial to mesenchymaltransitions (EMTs) (119 genes, FIG. 20). FIG. 20 shows genes marked withK79me2 specifically in fibroblasts, in ESCs and in both cell types.Genes that have 10-fold or more H3K79me2 in fibroblasts than in ES cellswere designated as Fibroblasts-specific K79me2 marked genes (upper leftdotted line). Genes that have 10-fold or more H3K79me2 in ES cells thanin fibroblasts were designated as ES-specific K79me2 marked genes (lowerright dotted line). Top 5 gene sets that overlap with these set of genesare indicated in the boxes. Interestingly, it was observed that 6 Daysafter OSKM expression, 143 genes had lost H3K79me2 2-fold or more (FIG.5A). FIG. 5 shows genome-wide analysis of H3K79me2 marks duringreprogramming. FIG. 5A shows genome-wide representation of H3K79me2marked genes in fibroblasts and in fibroblasts 6 days intoreprogramming. Each dot represents a gene and the shade indicates theH3K79me2 enrichment of that gene in ES cells. The dotted line indicatesthe set of genes that lose K79me2 2-fold or more upon OSKM transduction.The top 5 gene sets that overlap with these genes are indicated in thebox below. Gene set overlap analysis indicated that these 143 genes werealso highly significantly represented in gene sets associated with EMTphenotypes. Only a few of these genes had already decreased inexpression at Day 6 (9 out of 143), but a majority of them would losethis mark in the pluripotent state (115 out of 143 devoid of H3K79me2 inES cells). This finding lead to the question whether Dot1L inhibitionpromotes the removal of K79me2 from such fibroblast specific,EMT-associated genes. To explore this notion, ChIP-seq for K79me2 onOSKM expressing fibroblasts treated with iDot1L was performed. It wasobserved that upon inhibitor treatment, K79me2 levels were reduced onalmost all genes with the exception of a subset that comprised mostly ofhousekeeping genes. This subset of genes also had high levels of K79me2in ES cells indicating that these active loci turnover H3K79me2 slowly(FIG. 5B). FIG. 5B shows genome-wide representation of H3K79me2 markedgenes in fibroblasts and in fibroblasts treated with iDot1L 6 days intoreprogramming. Each dot represents a gene and the shade indicates theH3K79me2 enrichment of that gene in ES cells. Note that genes that havehigh K79me2 in ES cells retain this mark despite Dot1L inhibition. Thedotted line indicates the set of genes that lose K79me2 10-fold or morein iDot1L-treated fibroblasts. The top 5 gene sets that overlap withthese genes are indicated in the box below. Strikingly, the genes thatlost proportionally the most K79me2 in inhibitor-treated fibroblastsduring reprogramming compared to the initial fibroblasts were againhighly significantly represented in gene sets associated with EMTs (FIG.5B). In fact, master regulators of mesenchymal states such as Zeb1/2,Snai1/2, Grem1 and TGFB2, were among these genes (FIG. 5C)¹⁹. FIG. 5Cshows H3K79me2 ChIP-sequencing tracks for select EMT-associated genes infibroblasts and H3K27me3 in ES cells.

These observations support the notion that Dot1L inhibition collaborateswith OSKM in facilitating the loss of K79me2 from fibroblast specificregulators.

Methods Summary

shRNAs were designed using the RNAi Codex²⁶. 97-mer oligonucleotides(Table 2) were PCR amplified and cloned into MSCV-PM²⁷ vector. ControlshRNA targeting the firefly luciferase²⁷ and the Nanog shRNA werepreviously described²⁸. Reprogramming assays were carried out witheither retroviral⁷ or lentiviral²⁹ reprogramming factors. dH1f cellswere previously described³. IMR-90 and MRC5 human diploid fibroblastswere purchased from ATCC. Immunostaining of reprogramming plates wereperformed as described⁸. For gene expression analyses, total RNA wasextracted from three independent culture plates for each condition andtranscriptional profiling was performed using Affymetrix U133Amicroarrays. Primers used for quantitative real-time PCR can be found inTable 3. ChIP-seq was performed as described with slightmodifications²⁵.

REFERENCES

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The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference in their entirety, particularly for the use or subjectmatter referenced herein.

What is claimed is:
 1. A method of producing induced pluripotent stemcells, the method comprising inhibiting Dot1L in a differentiated celland culturing the differentiated cell under reprogramming conditions toproduce induced pluripotent stem cells.
 2. The method of claim 1,wherein inhibiting Dot1L comprises inhibiting the methyltransferaseactivity of Dot1L.
 3. The method of claim 1, wherein Dot1L is inhibitedby contacting the differentiated cell with a composition comprising acompound of formula I, II, III, or IV.
 4. The method of claim 1, whereinDot1L is inhibited by contacting the differentiated cell with acomposition comprising a compound of formula:


5. The method of claim 1, wherein Dot1L is inhibited by contacting thedifferentiated cell with an shRNA that knocks down Dot1L expression. 6.The method of claim 1, wherein the reprogramming conditions comprise areprogramming cocktail comprising a transcription factor.
 7. The methodof claim 6, wherein the reprogramming cocktail comprises Oct4 and Sox2.8. (canceled)
 9. The method of claim 6, wherein the reprogrammingcocktail does not include Klf4 or c-Myc.
 10. The method of claim 6,wherein Dot1L is inhibited when the cell is cultured with thereprogramming cocktail.
 11. The method of claim 1, wherein thedifferentiated cell is a fibroblast.
 12. (canceled)
 13. The method ofclaim 1, wherein the differentiated cell is a human cell.
 14. The methodof claim 13, wherein the differentiated human cell is dH1fs, IMR-90 orMRC-5.
 15. The method of claim 1, wherein the differentiated cell is amouse cell. 16-19. (canceled)
 20. A method of accelerating theproduction of induced pluripotent stem cells, the method comprisinginhibiting Dot1L in a differentiated cell and culturing thedifferentiated cell under reprogramming conditions to accelerate theproduction of induced pluripotent stem cells, wherein the production ofinduced pluripotent stem cells is accelerated compared to adifferentiated cell in which Dot1L is not inhibited.
 21. A method ofproducing induced pluripotent stem cells, the method comprisingupregulating the expression of Nanog and Lin28 in a differentiated celland culturing the differentiated cell under reprogramming conditions toproduce induced pluripotent stem cells.
 22. The method of claim 21,wherein the expression of Nanog and Lin28 is upregulated by inhibitingDot1L.
 23. A method of producing induced pluripotent stem cells, themethod comprising inhibiting SUV39H1 in a differentiated cell andculturing the differentiated cell under reprogramming conditions toproduce induced pluripotent stem cells.
 24. A method of producinginduced pluripotent stem cells, the method comprising inhibiting YY1 ina differentiated cell and culturing the differentiated cell underreprogramming conditions to produce induced pluripotent stem cells. 25.A composition comprising a population of human induced pluripotent stemcells produced according to the method of claim
 1. 26. (canceled)
 27. Amethod of treatment comprising administering to a person in need thereofthe composition of claim
 25. 28. (canceled)